Nucleic acid and corresponding protein entitled 161P2F10B useful in treatment and detection of cancer

ABSTRACT

A gene (designated 161P2F10B) and its encoded protein are described wherein 161P2F10B exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed in the cancers listed in Table I. Consequently, 161P2F10B provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 161P2F10B gene or fragment thereof, or its encoded protein or a fragment thereof, can be used to elicit a humoral or cellular immune response.

RELATED APPLICATION

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/062,109, filed Jan. 31, 2002, which is a continuation ofU.S. patent application Ser. No. 10/005,480, filed Nov. 7, 2001, nowabandoned. The contents of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION ON COMPACT DISC

The content of the following submission on compact discs is incorporatedherein by reference in its entirety: A compact disc copy of the SequenceListing (COPY 1) (file name: 511582006204, date recorded: Mar. 31, 2005,size: 169,984 bytes); and a duplicate compact disc copy of the SequenceListing (COPY 2) (file name: 511582006204, date recorded: Mar. 31, 2005,size: 169,984 bytes).

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encodedprotein, termed 161P2F10B, expressed in certain cancers, and todiagnostic and therapeutic methods and compositions useful in themanagement of cancers that express 161P2F10B.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl AcadSci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

As will be discussed in detail below, the gene and corresponding proteinreferred to as 161P2F10B is identical to ENPP3 phosphodiesterase (alsocalled CD203c or PD-1 beta). ENPP3 is an ecto-enzyme belonging to afamily of ectonucleotide phosphodiesterases and pyrophosphatases. ENPP3is a phosphodiesterase I ecto-enzyme. It is expressed in normal prostateand uterus, as well as on basophils and mast cells. Expression on thehematopoietic cells is upregulated in presence of allergen or bycross-linking with IgE (Buring et al., 1999, Blood 94: 2343). Members ofthe ENPP family possess ATPase and ATP pyrophosphatase activities. Theyhydrolyze extracellular nucleotides, nucleoside phosphates, and NAD.They are involved in extracellular nucleotide metabolism, nucleotidesignaling, and recycling of extracellular nucleotides. They are alsoinvolved in cell-cell and cell-matrix interactions. ENPP enzymes differin their substrate specificity and tissue distribution. ENPP enzymesalso play a role in recycling extracellular nucleotides. It has beendemonstrated that ENNP1 allows activated T-cells to use NAD+ from dyingcells as a source of adenosine. ENPP3 expressed in the intestine mayalso be involved in the hydrolysis of nucleotides derived from food(Byrd et al 1985, Scott et al. 1997).

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 161P2F10B, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 161P2F10B gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of161P2F10B are provided. The tissue-related profile of 161P2F10B innormal adult tissues, combined with the over-expression observed in thetumors listed in Table I, shows that 161P2F10B is aberrantlyover-expressed in at least some cancers, and thus serves as a usefuldiagnostic, prophylactic, prognostic, and/or therapeutic target forcancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 161P2F10B genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding161P2F10B-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a161P2F10B-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the161P2F10B genes or mRNA sequences or parts thereof, and polynucleotidesor oligonucleotides that hybridize to the 161P2F10B genes, mRNAs, or to161P2F10B-encoding polynucleotides. Also provided are means forisolating cDNAs and the genes encoding 161P2F10B. Recombinant DNAmolecules containing 161P2F10B polynucleotides, cells transformed ortransduced with such molecules, and host-vector systems for theexpression of 161P2F10B gene products are also provided. The inventionfurther provides antibodies that bind to 161P2F10B proteins andpolypeptide fragments thereof, including polyclonal and monoclonalantibodies, murine and other mammalian antibodies, chimeric antibodies,humanized and fully human antibodies, and antibodies labeled with adetectable marker or therapeutic agent. In certain embodiments there isa proviso that the entire nucleic acid sequence of FIG. 2 is not encodedand/or the entire amino acid sequence of FIG. 2 is not prepared. Incertain embodiments, the entire nucleic acid sequence of FIG. 2 isencoded and/or the entire amino acid sequence of FIG. 2 is prepared,either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 161P2F10B polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express161P2F10B. A typical embodiment of this invention provides methods formonitoring 161P2F10B gene products in a tissue or hematology samplehaving or suspected of having some form of growth dysregulation such ascancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 161P2F10Bsuch as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of161P2F10B as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 161P2F10B in a human subject wherein thecomposition comprises a carrier suitable for human use and a human unitdose of one or more than one agent that inhibits the production orfunction of 161P2F10B. Preferably, the carrier is a uniquely humancarrier. In another aspect of the invention, the agent is a moiety thatis immunoreactive with 161P2F10B protein. Non-limiting examples of suchmoieties include, but are not limited to, antibodies (such as singlechain, monoclonal, polyclonal, humanized, chimeric, or humanantibodies), functional equivalents thereof (whether naturally occurringor synthetic), and combinations thereof. The antibodies can beconjugated to a diagnostic or therapeutic moiety. In another aspect, theagent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 161P2F10B and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 161P2F10B as describedabove. The one or more than one nucleic acid molecule may also be, orencodes, a molecule that inhibits production of 161P2F10B. Non-limitingexamples of such molecules include, but are not limited to, thosecomplementary to a nucleotide sequence essential for production of161P2F10B (e.g. antisense sequences or molecules that form a triplehelix with a nucleotide double helix essential for 161P2F10B production)or a ribozyme effective to lyse 161P2F10B mRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 161P2F10B SSH sequence.

FIG. 2. The cDNA and amino acid sequence of 161P2F10B (FIG. 2A), and thenucleic acid and amino acid sequence of 161P2F10B variant 1 (FIG. 2B).The codon for the start methionine is underlined. The open reading framefor each extends from nucleic acid 44 to 2671 including the stop codon

FIG. 3. Amino acid sequence of 161P2F10B (FIG. 3A) and the amino acidsequence of 161P2F10B variant 1 (FIG. 3B). Each protein has 875 aminoacids.

FIG. 4. FIG. 4A provides the amino acid alignment of 161P2F10B withENPP3;

FIG. 4B provides an amino acid alignment of 161P2F10B with 161P2F10Bvariant 1; FIG. 4C provides the Alignment of 161P2F10B and SNP variant 2carrying a T to P mutation at position 874.

FIG. 5. Hydrophilicity amino acid profile of 161P2F10B determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website through the ExPasymolecular biology server.

FIG. 6. Hydropathicity amino acid profile of 161P2F10B determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website through the ExPasy molecular biologyserver.

FIG. 7. Percent accessible residues amino acid profile of 161P2F10Bdetermined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491492) accessed on the ProtScalewebsite through the ExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of 161P2F10B determinedby computer algorithm sequence analysis using the method of Bhaskaranand Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website through theExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of 161P2F10B determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed onthe ProtScale website through the ExPasy molecular biology server.

FIG. 10. Expression of 161P2F10B by RT-PCR. First strand cDNA wasprepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2(VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool,kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancerpool, breast cancer pool, metastasis cancer pool, pancreas cancer pool,and prostate cancer metastasis to lymph node from two differentpatients. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to 161P2F10B, was performedat 26 and 30 cycles of amplification. Strong expression of 161P2F10B wasobserved in kidney cancer pool. Expression was also detected in VP1,prostate cancer xenograft pool, prostate cancer pool and colon cancerpool. Low expression was observed in VP2, lung cancer pool, ovary cancerpool, breast cancer pool, metastasis pool, pancreas cancer pool, and inthe two different prostate cancer metastasis to lymph node.

FIG. 11. Expression of 161P2F10B in normal human tissues. Two multipletissue Northern blots, with 2 mg of mRNA/lane, were probed with the161P2F10B sequence. Size standards in kilobases (kb) are indicated onthe side. The results show expression of two 161P2F10B transcriptscomigrating at approximately 4.4 kb, in kidney, prostate and colon, andto lower levels, in thymus.

FIG. 12. Expression of 161P2F10B in kidney cancer xenografts. RNA wasextracted from normal kidney (N), prostate cancer xenografts, LAPC-4AD,LAPC-4AI, LAPC-9AD, and LAPC-9AI, and two kidney cancer xenografts (KiXeno-1 and Ki Xeno-2). A Northern blot with 10 mg of total RNA/lane wasprobed with the 161P2F10B sequence. Size standards in kilobases (kb) areindicated on the side. The results showed expression of 161P2F10B inboth kidney xenografts, LAPC-4AI, LAPC-9AI, but not in normal kidney orthe tested cell lines.

FIG. 13. Expression of 161P2F10B in patient kidney cancer specimens andin normal tissues. RNA was extracted from a pool of three kidneycancers, as well as from normal prostate (NP), normal bladder (NB),normal kidney (NK), and normal colon (NC). A Northern blot with 10 mg oftotal RNA/lane was probed with the 161P2F10B sequence. Size standards inkilobases (kb) are indicated on the side. The results showed expressionof 161P2F10B in the kidney cancer pool but not in the normal tissuestested.

FIG. 14. Expression of 161P2F10B in kidney cancer patient specimens. RNAwas extracted from kidney cancer cell lines (CL), normal kidney (N),kidney tumors (T), and matched normal adjacent tissue (NAT) isolatedfrom kidney cancer patients. Northern blots with 10 mg of total RNA/lanewere probed with the 161P2F10B sequence. Size standards in kilobases(kb) are indicated on the side. The results showed expression of161P2F10B in all four clear cell carcinoma kidney tumors, but not inpapillary carcinoma nor in normal kidney tissues.

FIG. 15. Expression of 161P2F10B in kidney cancer metastasis specimensand in normal tissues. RNA was extracted from kidney cancer metastasisto lung, kidney cancer metastasis to lymph node, normal bladder (NB),normal kidney (NK), and normal lung (NL), normal breast (NBr), normalovary (NO), and normal pancreas (NPa). Northern blots with 10 mg oftotal RNA/lane were probed with the 161P2F10B sequence. Size standardsin kilobases (kb) are indicated on the side. The results showedexpression of 161P2F10B in the two kidney cancer metastasis tested. Weakexpression was detected in normal kidney and normal breast but not inother normal tissues, but not in normal tissues. The ethidium-bromidestaining of the gel showed equivalent loading of the RNA samples.

FIG. 16: Detection of 161P2F10B protein by immunohistochemistry inkidney cancer patient specimens. Renal clear cell carcinoma tissue andits matched normal adjacent tissue as well as its metastatic cancer tolymph node were obtained from a kidney cancer patient. Frozen tissueswere cut into 4 micron sections and fixed in acetone for 10 minutes. Thesections were then incubated with PE-labeled mouse monoclonal anti-ENPP3antibody (Coulter-Immunotech, Marseilles, France) for 3 hours (FIG. 16panels A-F), or isotype control antibody (FIG. 16 panels G-I). Theslides were washed three times in buffer, and either analyzed byfluorescence microscopy (FIG. 16 panels A, B and C), or furtherincubated with DAKO EnVision+™ peroxidase-conjugated goat anti-mousesecondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour(FIG. 16 panels D, E, and F). The sections were then washed in buffer,developed using the DAB kit (SIGMA Chemicals), counterstained usinghematoxylin, and analyzed by bright field microscopy (FIG. 16 panels D,E and F). The results showed strong expression of 161P2F10B in the renalcarcinoma patient tissue (FIG. 16 panels A and D) and the kidney cancermetastasis to lymph node tissue (FIG. 16 panels C and F), but weakly innormal kidney (FIG. 16 B and E). The expression was detected mostlyaround the cell membrane indicating that 161P2F10B is membraneassociated in kidney cancer tissues. The weak expression detected innormal kidney was localized to the kidney tubules. The sections stainedwith the isotype control antibody were negative showing the specificityof the anti-ENPP3 antibody (FIG. 16 panels G-I).

FIG. 17: Expression of 161P2F10B protein on the cell surface of renalcell carcinoma xenografts. Renal cell carcinoma xenograft tissues (FIG.17 A) and renal cell carcinoma metastasis to lymph node xenografttissues (FIG. 17 B) were harvested from animals and dispersed intosingle cell suspension. The cells were stained using the commerciallyavailable antibody 97A6 specific for ENPP3 protein (also calledanti-CD203c) (Immunotech, Marseilles, France). They were then washed inPBS and analyzed by flow cytometry. The results showed strong expressionof 161P2F10B in both renal cell carcinoma xenograft (FIG. 17 A) as wellas renal cancer metastasis xenograft (FIG. 17 B). These data demonstratethat 161P2F10B is expressed on the cell surface of the kidney cancer andkidney cancer metastasis xenograft cells.

FIG. 18. Detection of 161P2F10B protein by immunohistochemistry in humancancer xenograft tissues. Renal cell carcinoma (FIG. 18 panels A, D, G),renal cell carcinoma metastasis to lymph node (FIG. 18 panels B, E, H),and prostate cancer LAPC-4AI (FIG. 18 panels C, F, I) xenografts weregrown in SCID mice. Xenograft tissues were harvested, 4 micron thickfrozen sections were cut and fixed in acetone for 10 minutes. Thesections were then incubated with PE-labeled mouse monoclonal anti-ENPP3antibody (Immunotech, Marseilles, France) for 3 hours (FIG. 18 panelsA-F), or isotype control antibody (FIG. 18 panels G-I). The slides werewashed three times in buffer, and either analyzed by fluorescencemicroscopy (FIG. 18 panels A-C), or further incubated with DAKOEnVision+™ peroxidase-conjugated goat anti-mouse secondary antibody(DAKO Corporation, Carpenteria, Calif.) for 1 hour (FIG. 18 panels D-I).The sections were then washed in buffer, developed using the DAB kit(SIGMA Chemicals), counterstained using hematoxylin, and analyzed bybright field microscopy (FIG. 18 panels C-F). The results showed strongexpression of 161P2F10B in the renal cell carcinoma xenograft tissue(FIG. 18 panels A and D), in the kidney cancer metastasis to lymph node(FIG. 18 panels B and E) as well as in the LAPC-4AI prostate xenograft(C and F) but not in the negative isotype control sections (FIG. 18panels G, H, I). The expression was detected mostly around the cellmembrane indicating that 161P2F10B is membrane-associated.

FIG. 19. The secondary structure of 161P2F10B, namely the predictedpresence and location of alpha helices, extended strands, and randomcoils, is predicted from the primary amino acid sequence using theHNN—Hierarchical Neural Network method (Guermeur, 1997), accessed fromthe ExPasy molecular biology server. The analysis indicates that161P2F10B is composed 31.31% alpha helix, 11.31% extended strand, and57.37% random coil (FIG. 19A). Shown graphically in FIG. 19 panels B andC are the results of analysis using the TMpred (FIG. 19B) and TMHMM(FIG. 19C) prediction programs depicting the location of thetransmembrane domain.

FIG. 20. Expression of 161P2F10B in Human Patient Cancers by WesternBlot. Cell lysates from kidney cancer tissues (KiCa), kidney cancermetastasis to lymph node (KiCa Met), as well as normal kidney (NK) weresubjected to Western analysis using an anti-161P2F10B mouse monoclonalantibody. Briefly, tissues (˜25 μg total protein) were solubilized inSDS-PAGE sample buffer and separated on a 10-20% SDS-PAGE gel andtransferred to nitrocellulose. Blots were blocked in Tris-bufferedsaline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk. Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa. These resultsdemonstrate that 161P2F10B may be useful as a diagnostic and therapeutictarget for kidney cancers, metastatic cancers and potentially otherhuman cancers.

FIG. 21. Expression of 161P2F10B in Human Xenograft Tissues by WesternBlot. Cell lysates from kidney cancer xenograft (KiCa Xeno), kidneycancer metastasis to lymph node xenograft (Met Xeno), as well as normalkidney (NK) were subjected to Western analysis using an anti-161P2F10Bmouse monoclonal antibody. Briefly, tissues (˜25 μg total protein) weresolubilized in SDS-PAGE sample buffer and separated on a 10-20% SDS-PAGEgel and transferred to nitrocellulose. Blots were blocked inTris-buffered saline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk. Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa, and a multimerof approximately 260 kDa. These results demonstrate that the humancancer xenograft mouse models can be used to study the diagnostic andtherapeutic effects of 161P2F10B.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 161P2F10B Polynucleotides

-   -   II.A.) Uses of 161P2F10B Polynucleotides        -   II.A.1.) Monitoring of Genetic Abnormalities        -   II.A.2.) Antisense Embodiments        -   II.A.3.) Primers and Primer Pairs        -   II.A.4.) Isolation of 161P2F10B-Encoding Nucleic Acid            Molecules        -   II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector            Systems

III.) 161P2F10B-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments    -   III.B.) Expression of 161P2F10B-related Proteins    -   III.C.) Modifications of 161P2F10B-related Proteins    -   III.D.) Uses of 161P2F10B-related Proteins

IV.) 161P2F10B Antibodies

V.) 161P2F10B Cellular Immune Responses

VI.) 161P2F10B Transgenic Animals

VII.) Methods for the Detection of 161P2F10B

VIII.) Methods for Monitoring the Status of 161P2F10B-related Genes andTheir Products

IX.) Identification of Molecules that Interact with 161P2F10B

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines    -   X.B.) 161P2F10B as a Target for Antibody-Based Therapy    -   X.C.) 161P2F10B as a Target for Cellular Immune Responses        -   X.C.1. Minigene Vaccines        -   X.C.2. Combinations of CTL Peptides with Helper Peptides        -   X.C.3. Combinations of CTL Peptides with T Cell Priming            Agents        -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL            and/or HTL Peptides    -   X.D.) Adoptive Immunotherapy    -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic        Purposes

XI.) Diagnostic and Prognostic Embodiments of 161P2F10B.

XII.) Inhibition of 161P2F10B Protein Function

-   -   XII.A.) Inhibition of 161P2F10B With Intracellular Antibodies    -   XII.B.) Inhibition of 161P2F10B with Recombinant Proteins    -   XII.C.) Inhibition of 161P2F10B Transcription or Translation    -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) Kits

I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 161P2F10B (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 161P2F10B. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a161P2F10B-related protein). For example an analog of the 161P2F10Bprotein can be specifically bound by an antibody or T cell thatspecifically binds to 161P2F10B.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-161P2F110B antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-161P2F10B antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-161P2F 110B antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1× SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 161P2F10B geneor that encode polypeptides other than 161P2F10B gene product orfragments thereof. A skilled artisan can readily employ nucleic acidisolation procedures to obtain an isolated 161P2F10B polynucleotide. Aprotein is said to be “isolated,” for example, when physical, mechanicalor chemical methods are employed to remove the 161P2F10B protein fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 161P2F10B protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage TxNxM+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 161P2F10B-related protein,refers to any pattern of amino acids forming part of the primarysequence of a protein, that is associated with a particular function(e.g. protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) can also be uracil (U); this definition pertainsto the differences between the chemical structures of DNA and RNA, inparticular the observation that one of the four major bases in RNA isuracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment, for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 161P2F10B, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 161P2F10B protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 161P2F10B protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm.DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 161P2F10B protein shown in FIG. 2 or FIG.3). An analog is an example of a variant protein.

The 161P2F10B-related proteins of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 161P2F10B proteins orfragments thereof, as well as fusion proteins of a 161P2F10B protein anda heterologous polypeptide are also included. Such 161P2F10B proteinsare collectively referred to as the 161P2F10B-related proteins, theproteins of the invention, or 161P2F10B. The term “161P2F10B-relatedprotein” refers to a polypeptide fragment or an 161P2F10B proteinsequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30,35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100amino acids.

II.) 161P2F10B Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 161P2F10B gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 161P2F10B-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 161P2F10B gene or mRNA sequence ora part thereof, and polynucleotides or oligonucleotides that hybridizeto an 161P2F10B gene, mRNA, or to an 161P2F10B encoding polynucleotide(collectively, “161P2F10B polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 161P2F10B polynucleotide include: a 161P2F10Bpolynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 161P2F10B as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 161P2F10B nucleotides comprise, without limitation:

-   -   (a) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2, wherein T can also be U;    -   (b) a polynucleotide comprising or consisting of the sequence as        shown in FIG. 2, from nucleotide residue number 44 through        nucleotide residue number 2671, wherein T can also be U;    -   (c) a polynucleotide that encodes an 161P2F10B-related protein        that is at least 90% homologous to the entire amino acid        sequence shown in FIG. 2;    -   (d) a polynucleotide that encodes an 161P2F10B-related protein        that is at least 90% identical to the entire amino acid sequence        shown in FIG. 2;    -   (e) a polynucleotide that encodes at least one peptide set forth        in Tables V-XVIII;    -   (f) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 875        that includes an amino acid position having a value greater than        0.5 in the Hydrophilicity profile of FIG. 5;    -   (g) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 875        that includes an amino acid position having a value less than        0.5 in the Hydropathicity profile of FIG. 6;    -   (h) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 875        that includes an amino acid position having a value greater than        0.5 in the Percent Accessible Residues profile of FIG. 7;    -   (i) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 875        that includes an amino acid position having a value greater than        0.5 in the Average Flexibility profile on FIG. 8;    -   (j) a polynucleotide that encodes a peptide region of at least 5        amino acids of FIG. 3 in any whole number increment up to 875        that includes an amino acid position having a value greater than        0.5 in the Beta-turn profile of FIG. 9;    -   (k) a polynucleotide that is fully complementary to a        polynucleotide of any one of    -   (l) a polynucleotide that selectively hybridizes under stringent        conditions to a polynucleotide of (a)-(k);    -   (m) a peptide that is encoded by any of (a)-(j); and,    -   (n) a polynucleotide of any of (a)-(l) or peptide of (m)        together with a pharmaceutical excipient and/or in a human unit        dose form.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 161P2F10Bpolynucleotides that encode specific portions of the 161P2F10B mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, 725, 750, 775, 900, 825, 850, or 875 contiguous amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 161P2F10B protein shown in FIG. 2,or FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 161P2F10B protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 40 to about amino acid 50 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 50 to about amino acid 60 of the 161P2F10B protein shown in FIG. 2or FIG. 3, polynucleotides encoding about amino acid 60 to about aminoacid 70 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 70 to about amino acid 80 ofthe 161P2F10B protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 80 to about amino acid 90 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 90 to about amino acid 100 of the 161P2F10B protein shown in FIG. 2or FIG. 3, in increments of about 10 amino acids, ending at the carboxylterminal amino acid set forth in FIG. 2 or FIG. 3. Accordinglypolynucleotides encoding portions of the amino acid sequence (of about10 amino acids), of amino acids 100 through the carboxyl terminal aminoacid of the 161P2F10B protein are embodiments of the invention. Whereinit is understood that each particular amino acid position discloses thatposition plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of the 161P2F10Bprotein are also within the scope of the invention. For example,polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the161P2F10B00 protein shown in FIG. 2 or FIG. 3 can be generated by avariety of techniques well known in the art. These polynucleotidefragments can include any portion of the 161P2F10B sequence as shown inFIG. 2 or FIG. 3.

Additional illustrative embodiments of the invention disclosed hereininclude 161P2F10B polynucleotide fragments encoding one or more of thebiological motifs contained within the 161P2F10B protein sequence,including one or more of the motif-bearing subsequences of the 161P2F10Bprotein set forth in Tables V-XVIII. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 161P2F100B that exhibit homology to a known molecule. Inanother embodiment of the invention, typical polynucleotide fragmentscan encode one or more of the 161P2F10B N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of 161P2F10B Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 161P2F10B gene maps to thechromosomal location set forth in Example 3. For example, because the161P2F10B gene maps to this chromosome, polynucleotides that encodedifferent regions of the 161P2F10B protein are used to characterizecytogenetic abnormalities of this chromosomal locale, such asabnormalities that are identified as being associated with variouscancers. In certain genes, a variety of chromosomal abnormalitiesincluding rearrangements have been identified as frequent cytogeneticabnormalities in a number of different cancers (see e.g. Krajinovic etal., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10):3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)).Thus, polynucleotides encoding specific regions of the 161P2F10B proteinprovide new tools that can be used to delineate, with greater precisionthan previously possible, cytogenetic abnormalities in the chromosomalregion that encodes 161P2F10B that may contribute to the malignantphenotype. In this context, these polynucleotides satisfy a need in theart for expanding the sensitivity of chromosomal screening in order toidentify more subtle and less common chromosomal abnormalities (see e.g.Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 161P2F10B was shown to be highly expressed in prostateand other cancers, 161P2F10B polynucleotides are used in methodsassessing the status of 161P2F10B gene products in normal versuscancerous tissues. Typically, polynucleotides that encode specificregions of the 161P2F10B protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations, oralterations resulting in a loss of an antigen etc.) in specific regionsof the 161P2F10B gene, such as such regions containing one or moremotifs. Exemplary assays include both RT-PCR assays as well assingle-strand conformation polymorphism (SSCP) analysis (see, e.g.,Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of whichutilize polynucleotides encoding specific regions of a protein toexamine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 161P2F10B. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 161P2F10B polynucleotidesand polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,161P2F10B. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 161P2F10B antisense oligonucleotides of the presentinvention include derivatives such as S-oligonucleotides(phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra),which exhibit enhanced cancer cell growth inhibitory action. S-oligos(nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention can be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfurtransfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 161P2F10B antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The 161P2F10B antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of the161P2F10B genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 161P2F10B mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 161P2F10B antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 161P2F10B mRNA. Optionally, 161P2F10Bantisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 161P2F10B. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 161P2F10B expression, see, e.g.,L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 161P2F10B polynucleotide in a sample and as ameans for detecting a cell expressing a 161P2F10B protein.

Examples of such probes include polypeptides comprising all or part ofthe human 161P2F10B cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 161P2F10B mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 161P2F10B mRNA.

The 161P2F10B polynucleotides of the invention are useful for a varietyof purposes, including but not limited to their use as probes andprimers for the amplification and/or detection of the 161P2F10B gene(s),mRNA(s), or fragments thereof; as reagents for the diagnosis and/orprognosis of prostate cancer and other cancers; as coding sequencescapable of directing the expression of 161P2F10B polypeptides; as toolsfor modulating or inhibiting the expression of the 161P2F10B gene(s)and/or translation of the 161P2F10B transcript(s); and as therapeuticagents.

The present invention includes the use of any probe as described hereinto identify and isolate a 161P2F10B or 161P2F10B related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 161P2F10B-Encoding Nucleic Acid Molecules

The 161P2F10B cDNA sequences described herein enable the isolation ofother polynucleotides encoding 161P2F10B gene product(s), as well as theisolation of polynucleotides encoding 161P2F10B gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 161P2F10B gene product as well as polynucleotides that encodeanalogs of 161P2F10B-related proteins. Various molecular cloning methodsthat can be employed to isolate full length cDNAs encoding an 161P2F10Bgene are well known (see, for example, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, NewYork, 1989; Current Protocols in Molecular Biology. Ausubel et al.,Eds., Wiley and Sons, 1995). For example, lambda phage cloningmethodologies can be conveniently employed, using commercially availablecloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clonescontaining 161P2F10B gene cDNAs can be identified by probing with alabeled 161P2F10B cDNA or a fragment thereof. For example, in oneembodiment, the 161P2F10B cDNA (FIG. 2) or a portion thereof can besynthesized and used as a probe to retrieve overlapping and full-lengthcDNAs corresponding to a 161P2F10B gene. The 161P2F10B gene itself canbe isolated by screening genomic DNA libraries, bacterial artificialchromosome libraries (BACs), yeast artificial chromosome libraries(YACs), and the like, with 161P2F10B DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 161P2F10B polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 161P2F10B polynucleotide,fragment, analog or homologue thereof within a suitable prokaryotic oreukaryotic host cell. Examples of suitable eukaryotic host cells includea yeast cell, a plant cell, or an animal cell, such as a mammalian cellor an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of161P2F10B or a fragment, analog or homolog thereof can be used togenerate 161P2F10B proteins or fragments thereof using any number ofhost-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of161P2F10B proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 161P2F10B can be expressed in several prostatecancer and non-prostate cell lines, including for example 293, 293T,rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention areuseful for the production of a 161P2F10B protein or fragment thereof.Such host-vector systems can be employed to study the functionalproperties of 161P2F10B and 161P2F10B mutations or analogs.

Recombinant human 161P2F10B protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 161P2F10B-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 161P2F10B or fragment,analog or homolog thereof, the 161P2F10B or related protein is expressedin the 293T cells, and the recombinant 161P2F10B protein is isolatedusing standard purification methods (e.g., affinity purification usinganti-161P2F10B antibodies). In another embodiment, a 161P2F10B codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 161P2F10B expressing cell lines. Variousother expression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the161P2F10B coding sequence can be used for the generation of a secretedform of recombinant 161P2F10B protein.

As discussed herein, redundancy in the genetic code permits variation in161P2F10B gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 161P2F10B-Related Proteins

Another aspect of the present invention provides 161P2F10B-relatedproteins. Specific embodiments of 161P2F10B proteins comprise apolypeptide having all or part of the amino acid sequence of human161P2F10B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of161P2F10B proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 161P2F10B shown in FIG. 2or FIG. 3.

In general, naturally occurring allelic variants of human 161P2F10Bshare a high degree of structural identity and homology (e.g., 90% ormore homology). Typically, allelic variants of the 161P2F10B proteincontain conservative amino acid substitutions within the 161P2F10Bsequences described herein or contain a substitution of an amino acidfrom a corresponding position in a homologue of 161P2F10B. One class of161P2F10B allelic variants are proteins that share a high degree ofhomology with at least a small region of a particular 161P2F10B aminoacid sequence, but further contain a radical departure from thesequence, such as a non-conservative substitution, truncation, insertionor frame shift. In comparisons of protein sequences, the terms,similarity, identity, and homology each have a distinct meaning asappreciated in the field of genetics. Moreover, orthology and paralogycan be important concepts describing the relationship of members of agiven protein family in one organism to the members of the same familyin other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 161P2F10B proteins such aspolypeptides having amino acid insertions, deletions and substitutions.161P2F10B variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 161P2F10B variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 161P2F10B variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 161P2F10B protein having the amino acid sequence of SEQID NO: 703. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 161P2F10B variant alsospecifically binds to the 161P2F10B protein having the amino acidsequence of SEQ ID NO: 703. A polypeptide ceases to be a variant of theprotein shown in SEQ ID NO: 703 when it no longer contains any epitopecapable of being recognized by an antibody or T cell that specificallybinds to the 161P2F10B protein. Those skilled in the art understand thatantibodies that recognize proteins bind to epitopes of varying size, anda grouping of the order of about four or five amino acids, contiguous ornot, is regarded as a typical number of amino acids in a minimalepitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955;Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., JImmunol (1985) 135(4):2598-608.

Another class of 161P2F10B-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of FIG. 2 ora fragment thereof. Another specific class of 161P2F10B protein variantsor analogs comprise one or more of the 161P2F10B biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 161P2F10B fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the fill amino acid sequence of the161P2F10B protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the161P2F10B protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 20 to about amino acid 30 of the 161P2F10B proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid30 to about amino acid 40 of the 161P2F10B protein shown in FIG. 2 orFIG. 3, polypeptides consisting of about amino acid 40 to about aminoacid 50 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 50 to about amino acid 60 of the161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 60 to about amino acid 70 of the 161P2F10B proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid70 to about amino acid 80 of the 161P2F10B protein shown in FIG. 2 orFIG. 3, polypeptides consisting of about amino acid 80 to about aminoacid 90 of the 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 90 to about amino acid 100 of the161P2F10B protein shown in FIG. 2 or FIG. 3, etc. throughout theentirety of the 161P2F10B amino acid sequence. Moreover, polypeptidesconsisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 130, or 140 or 150 etc.) of the 161P2F10B protein shown inFIG. 2 or FIG. 3 are embodiments of the invention. It is to beappreciated that the starting and stopping positions in this paragraphrefer to the specified position as well as that position plus or minus 5residues.

161P2F10B-related proteins are generated using standard peptidesynthesis technology or using chemical cleavage methods well known inthe art. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a 161P2F10B-related protein. In oneembodiment, nucleic acid molecules provide a means to generate definedfragments of the 161P2F10B protein (or variants, homologs or analogsthereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 161P2F10B polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within the 161P2F10Bpolypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,Epimatrix™ and Epimer™, Brown University; and BIMAS.

Motif bearing subsequences of the 161P2F10B protein are set forth andidentified in Table XIX.

Table XX sets forth several frequently occurring motifs based on pfamsearches. The columns of Table XX list (1) motif name abbreviation, (2)percent identity found amongst the different member of the motif family,(3) motif name or description and (4) most common function; locationinformation is included if the motif is relevant for location.

Polypeptides comprising one or more of the 161P2F10B motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 161P2F10B motifsdiscussed above are associated with growth dysregulation and because161P2F10B is overexpressed in certain cancers (See, e.g., Table I).Casein kinase II, cAMP and camp-dependent protein kinase, and ProteinKinase C, for example, are enzymes known to be associated with thedevelopment of the malignant phenotype (see e.g. Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification also associated withcancer and cancer progression (see e.g. Treston et al., J. Natl. CancerInst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 161P2F10B protein that are capable of optimally binding to specifiedHLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University;and BIMAS). Moreover, processes for identifying peptides that havesufficient binding affinity for HLA molecules and which are correlatedwith being immunogenic epitopes, are well known in the art, and arecarried out without undue experimentation. In addition, processes foridentifying peptides that are immunogenic epitopes, are well known inthe art, and are carried out without undue experimentation either invitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

161P2F10B-related proteins are embodied in many forms, preferably inisolated form. A purified 161P2F10B protein molecule will besubstantially free of other proteins or molecules that impair thebinding of 161P2F10B to antibody, T cell or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 161P2F10B-related proteins include purified161P2F10B-related proteins and functional, soluble 161P2F10B-relatedproteins. In one embodiment, a functional, soluble 161P2F10B protein orfragment thereof retains the ability to be bound by antibody, T cell orother ligand.

The invention also provides 161P2F10B proteins comprising biologicallyactive fragments of the 161P2F10B amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the 161P2F10B protein, suchas the ability to elicit the generation of antibodies that specificallybind an epitope associated with the 161P2F10B protein; to be bound bysuch antibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL.

161P2F10B-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-161P2F10B antibodies, or T cells or in identifyingcellular factors that bind to 161P2F10B.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 161P2F10B protein that are capable of optimallybinding to specified HLA alleles (e.g., by using the SYFPEITHI site; thelistings in Table IV (A)-(E); Epimatrix™ and Epimer™, Brown University;and BIMAS. Illustrating this, peptide epitopes from 161P2F10B that arepresented in the context of human MHC class I molecules HLA-A1, A2, A3,A11, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, thecomplete amino acid sequence of the 161P2F10B protein was entered intothe HLA Peptide Motif Search algorithm found in the Bioinformatics andMolecular Analysis Section (BIMAS) web site listed above. The HLApeptide motif search algorithm was developed by Dr. Ken Parker based onbinding of specific peptide sequences in the groove of HLA Class Imolecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351:290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J.Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75(1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and10-mer peptides from a complete protein sequence for predicted bindingto HLA-A2 as well as numerous other HLA Class I molecules. Many HLAclass I binding peptides are 8-, 9-, 10 or 11-mers. For example, forclass I HLA-A2, the epitopes preferably contain a leucine (L) ormethionine (M) at position 2 and a valine (V) or leucine (L) at theC-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)).Selected results of 161P2F10B predicted binding peptides are shown inTables V-XVIII herein. In Tables V-XVIII, the top 50 ranking candidates,9-mers and 10-mers, for each family member are shown along with theirlocation, the amino acid sequence of each specific peptide, and anestimated binding score. The binding score corresponds to the estimatedhalf time of dissociation of complexes containing the peptide at 37° C.at pH 6.5. Peptides with the highest binding score are predicted to bethe most tightly bound to HLA Class I on the cell surface for thegreatest period of time and thus represent the best immunogenic targetsfor T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV are to be “applied” to the 161P2F10B protein. Asused in this context “applied” means that the 161P2F10B protein isevaluated, e.g., visually or by computer-based patterns finding methods,as appreciated by those of skill in the relevant art. Every subsequenceof the 161P2F10B of 8, 9, 10, or 11 amino acid residues that bears anHLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 161P2F10B-Related Proteins

In an embodiment described in the examples that follow, 161P2F10B can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 161P2F10B with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 161P2F10B protein intransfected cells. The secreted HIS-tagged 161P2F10B in the culturemedia can be purified, e.g., using a nickel column using standardtechniques.

III.C.) Modifications of 161P2F10B-Related Proteins

Modifications of 161P2F10B-related proteins such as covalentmodifications are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a 161P2F10B polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of the 161P2F10B. Another type of covalent modification of the161P2F10B polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of a protein of theinvention. Another type of covalent modification of 161P2F10B compriseslinking the 161P2F10B polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The 161P2F10B-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 161P2F10B fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 161P2F10B sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of161P2F10B. A chimeric molecule can comprise a fusion of a161P2F10B-related protein with a polyhistidine epitope tag, whichprovides an epitope to which immobilized nickel can selectively bind,with cytokines or with growth factors. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 161P2F10B. In analternative embodiment, the chimeric molecule can comprise a fusion of a161P2F10B-related protein with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 161P2F10B polypeptide in place of at least one variable regionwithin an Ig molecule. In a preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgGI molecule. For the production of immunoglobulinfusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 161P2F10B-Related Proteins

The proteins of the invention have a number of different specific uses.As 161P2F10B is highly expressed in prostate and other cancers,161P2F10B-related proteins are used in methods that assess the status of161P2F10B gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 161P2F10B protein are used to assess thepresence of perturbations (such as deletions, insertions, pointmutations etc.) in those regions (such as regions containing one or moremotifs). Exemplary assays utilize antibodies or T cells targeting161P2F10B-related proteins comprising the amino acid residues of one ormore of the biological motifs contained within the 161P2F10B polypeptidesequence in order to evaluate the characteristics of this region innormal versus cancerous tissues or to elicit an immune response to theepitope. Alternatively, 161P2F10B-related proteins that contain theamino acid residues of one or more of the biological motifs in the161P2F10B protein are used to screen for factors that interact with thatregion of 161P2F10B.

161P2F10B protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an161P2F10B protein), for identifying agents or cellular factors that bindto 161P2F10B or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 161P2F10B genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 161P2F10B gene product.Antibodies raised against an 161P2F10B protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 161P2F10Bprotein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 161P2F10B-related nucleic acids or proteins are also usedin generating HTL or CTL responses.

Various immunological assays useful for the detection of 161P2F10Bproteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 161P2F10B-expressingcells (e.g., in radioscintigraphic imaging methods). 161P2F10B proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 161P2F10B Antibodies

Another aspect of the invention provides antibodies that bind to161P2F10B-related proteins. Preferred antibodies specifically bind to a161P2F10B-related protein and do not bind (or bind weakly) to peptidesor proteins that are not 161P2F10B-related proteins. For example,antibodies that bind 161P2F10B can bind 161P2F10B-related proteins suchas the homologs or analogs thereof.

161P2F10B antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 161P2F10B isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of161P2F10B is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 161P2F10B and mutant 161P2F10B-relatedproteins. Such assays can comprise one or more 161P2F10B antibodiescapable of recognizing and binding a 161P2F10B-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 161P2F10B are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 161P2F10B antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 161P2F10Bexpressing cancers such as prostate cancer.

161P2F10B antibodies are also used in methods for purifying a161P2F10B-related protein and for isolating 161P2F10B homologues andrelated molecules. For example, a method of purifying a161P2F10B-related protein comprises incubating an 161P2F10B antibody,which has been coupled to a solid matrix, with a lysate or othersolution containing a 161P2F10B-related protein under conditions thatpermit the 161P2F10B antibody to bind to the 161P2F10B-related protein;washing the solid matrix to eliminate impurities; and eluting the161P2F10B-related protein from the coupled antibody. Other uses of the161P2F10B antibodies of the invention include generating anti-idiotypicantibodies that mimic the 161P2F10B protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 161P2F10B-related protein, peptide, or fragment,in isolated or immunoconjugated form (Antibodies: A Laboratory Manual,CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, ColdSpring Harbor Press, NY (1989)). In addition, fusion proteins of161P2F10B can also be used, such as a 161P2F10B GST-fusion protein. In aparticular embodiment, a GST fusion protein comprising all or most ofthe amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as animmunogen to generate appropriate antibodies. In another embodiment, a161P2F10B-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 161P2F10B-related protein or 161P2F10Bexpressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

The amino acid sequence of 161P2F10B as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 161P2F10B protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 161P2F10B amino acid sequence are used to identifyhydrophilic regions in the 161P2F110B structure. Regions of the161P2F10B protein that show immunogenic structure, as well as otherregions and domains, can readily be identified using various othermethods known in the art, such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Thus, each region identified by any of these programs or methods iswithin the scope of the present invention. Methods for the generation of161P2F10B antibodies are further illustrated by way of the examplesprovided herein. Methods for preparing a protein or polypeptide for useas an immunogen are well known in the art. Also well known in the artare methods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 161P2F10B immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

161P2F10B monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 161P2F10B-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 161P2F10B protein can also be produced in the context of chimericor complementarity determining region (CDR) grafted antibodies ofmultiple species origin. Humanized or human 161P2F10B antibodies canalso be produced, and are preferred for use in therapeutic contexts.Methods for humanizing murine and other non-human antibodies, bysubstituting one or more of the non-human antibody CDRs forcorresponding human antibody sequences, are well known (see for example,Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also,Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims etal., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 161P2F10B monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human161P2F10B monoclonal antibodies can also be produced using transgenicmice engineered to contain human immunoglobulin gene loci as describedin PCT Patent Application WO98/24893, Kucherlapati and Jakobovits etal., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin.Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec.2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No.6,114598 issued 5 Sep. 2000). This method avoids the in vitromanipulation required with phage display technology and efficientlyproduces high affinity authentic human antibodies.

Reactivity of 161P2F10B antibodies with an 161P2F10B-related protein canbe established by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,161P2F10B-related proteins, 161P2F10B-expressing cells or extractsthereof. A 161P2F10B antibody or fragment thereof can be labeled with adetectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 161P2F10B epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

V.) 161P2F10B Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI; Sette, A. and Sidney, J.Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994;Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996;Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 161P2F10B Transgenic Animals

Nucleic acids that encode a 161P2F10B-related protein can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 161P2F10B can be used to clone genomic DNA that encodes161P2F10B. The cloned genomic sequences can then be used to generatetransgenic animals containing cells that express DNA that encode161P2F10B. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr.1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically,particular cells would be targeted for 161P2F10B transgene incorporationwith tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 161P2F10Bcan be used to examine the effect of increased expression of DNA thatencodes 161P2F10B. Such animals can be used as tester animals forreagents thought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with thisaspect of the invention, an animal is treated with a reagent and areduced incidence of a pathological condition, compared to untreatedanimals that bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 161P2F10B can be used toconstruct a 161P2F10B “knock out” animal that has a defective or alteredgene encoding 161P2F10B as a result of homologous recombination betweenthe endogenous gene encoding 161P2F10B and altered genomic DNA encoding161P2F10B introduced into an embryonic cell of the animal. For example,cDNA that encodes 161P2F10B can be used to clone genomic DNA encoding161P2F10B in accordance with established techniques. A portion of thegenomic DNA encoding 161P2F10B can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 161P2F10B polypeptide.

VII.) Methods for the Detection of 161P2F10B

Another aspect of the present invention relates to methods for detecting161P2F10B polynucleotides and 161P2F10B-related proteins, as well asmethods for identifying a cell that expresses 161P2F10B. The expressionprofile of 161P2F10B makes it a diagnostic marker for metastasizeddisease. Accordingly, the status of 161P2F10B gene products providesinformation useful for predicting a variety of factors includingsusceptibility to advanced stage disease, rate of progression, and/ortumor aggressiveness. As discussed in detail herein, the status of161P2F10B gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of161P2F10B polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 161P2F10B polynucleotides include, for example, a161P2F10B gene or fragment thereof, 161P2F10B mRNA, alternative splicevariant 161P2F10B mRNAs, and recombinant DNA or RNA molecules thatcontain a 161P2F10B polynucleotide. A number of methods for amplifyingand/or detecting the presence of 161P2F10B polynucleotides are wellknown in the art and can be employed in the practice of this aspect ofthe invention.

In one embodiment, a method for detecting an 161P2F10B mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 161P2F10B polynucleotides as sense and antisense primers toamplify 161P2F10B cDNAs therein; and detecting the presence of theamplified 161P2F10B cDNA. Optionally, the sequence of the amplified161P2F10B cDNA can be determined.

In another embodiment, a method of detecting a 161P2F10B gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 161P2F10B polynucleotides assense and antisense primers; and detecting the presence of the amplified161P2F10B gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequence provided forthe 161P2F10B (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an161P2F10B protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 161P2F10B-related protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, a method of detecting the presence of a161P2F10B-related protein in a biological sample comprises firstcontacting the sample with a 161P2F10B antibody, a 161P2F10B-reactivefragment thereof, or a recombinant protein containing an antigen bindingregion of a 161P2F10B antibody; and then detecting the binding of161P2F10B-related protein in the sample.

Methods for identifying a cell that expresses 161P2F10B are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 161P2F10B gene comprises detecting the presenceof 161P2F10B mRNA in the cell. Methods for the detection of particularmRNAs in cells are well known and include, for example, hybridizationassays using complementary DNA probes (such as in situ hybridizationusing labeled 161P2F10B riboprobes, Northern blot and relatedtechniques) and various nucleic acid amplification assays (such asRT-PCR using complementary primers specific for 161P2F10B, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like). Alternatively, an assay for identifying acell that expresses a 161P2F10B gene comprises detecting the presence of161P2F10B-related protein in the cell or secreted by the cell. Variousmethods for the detection of proteins are well known in the art and areemployed for the detection of 161P2F10B-related proteins and cells thatexpress 161P2F10B-related proteins.

161P2F10B expression analysis is also useful as a tool for identifyingand evaluating agents that modulate 161P2F10B gene expression. Forexample, 161P2F10B expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 161P2F10Bexpression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 161P2F10B expression by RT-PCR, nucleic acid hybridization orantibody binding.

VIII.) Methods for Monitoring the Status of 161P2F10B-Related Genes andtheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant161P2F10B expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 161P2F10B ina biological sample of interest can be compared, for example, to thestatus of 161P2F10B in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 161P2F10B in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9; 376(2):306-14 and U.S. Pat. No. 5,837,501) to compare161P2F10B status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 161P2F10B expressing cells) as well as the level, andbiological activity of expressed gene products (such as 161P2F10B mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 161P2F10B comprises a change in the location of 161P2F10Band/or 161P2F10B expressing cells and/or an increase in 161P2F10B mRNAand/or protein expression.

161P2F10B status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 161P2F10Bgene and gene products are found, for example in Ausubel et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 161P2F10B in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in the 161P2F10B gene), Northern analysis and/or PCRanalysis of 161P2F10B mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 161P2F10B mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of161P2F10B proteins and/or associations of 161P2F10B proteins withpolypeptide binding partners). Detectable 161P2F10B polynucleotidesinclude, for example, a 161P2F10B gene or fragment thereof, 161P2F10BmRNA, alternative splice variants, 161P2F10B mRNAs, and recombinant DNAor RNA molecules containing a 161P2F10B polynucleotide.

The expression profile of 161P2F10B makes it a diagnostic marker forlocal and/or metastasized disease, and provides information on thegrowth or oncogenic potential of a biological sample. In particular, thestatus of 161P2F10B provides information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining 161P2F10B status and diagnosing cancers that express161P2F10B, such as cancers of the tissues listed in Table I. Forexample, because 161P2F10B mRNA is so highly expressed in prostate andother cancers relative to normal prostate tissue, assays that evaluatethe levels of 161P2F10B mRNA transcripts or proteins in a biologicalsample can be used to diagnose a disease associated with 161P2F10Bdysregulation, and can provide prognostic information useful in definingappropriate therapeutic options.

The expression status of 161P2F10B provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 161P2F10Bin biological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 161P2F10B in a biological sample canbe examined by a number of well-known procedures in the art. Forexample, the status of 161P2F10B in a biological sample taken from aspecific location in the body can be examined by evaluating the samplefor the presence or absence of 161P2F10B expressing cells (e.g. thosethat express 161P2F10B mRNAs or proteins). This examination can provideevidence of dysregulated cellular growth, for example, when161P2F10B-expressing cells are found in a biological sample that doesnot normally contain such cells (such as a lymph node), because suchalterations in the status of 161P2F10B in a biological sample are oftenassociated with dysregulated cellular growth. Specifically, oneindicator of dysregulated cellular growth is the metastases of cancercells from an organ of origin (such as the prostate) to a different areaof the body (such as a lymph node). In this context, evidence ofdysregulated cellular growth is important for example because occultlymph node metastases can be detected in a substantial proportion ofpatients with prostate cancer, and such metastases are associated withknown predictors of disease progression (see, e.g., Murphy et al.,Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1):17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 161P2F10Bgene products by determining the status of 161P2F10B gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 161P2F10Bgene products in a corresponding normal sample. The presence of aberrant161P2F10B gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 161P2F10B mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 161P2F10B mRNA can, for example,be evaluated in tissue samples including but not limited to those listedin Table I. The presence of significant 161P2F10B expression in any ofthese tissues is useful to indicate the emergence, presence and/orseverity of a cancer, since the corresponding normal tissues do notexpress 161P2F10B mRNA or express it at lower levels.

In a related embodiment, 161P2F10B status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 161P2F10B protein expressed by cellsin a test tissue sample and comparing the level so determined to thelevel of 161P2F10B expressed in a corresponding normal sample. In oneembodiment, the presence of 161P2F10B protein is evaluated, for example,using immunohistochemical methods. 161P2F10B antibodies or bindingpartners capable of detecting 161P2F10B protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 161P2F10Bnucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 161P2F10B may be indicative of the presenceor promotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 161P2F10B indicates a potentialloss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 161P2F10B geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 161P2F10Bgene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-1 tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). A variety of assays for examining methylation status ofa gene are well known in the art. For example, one can utilize, inSouthern hybridization approaches, methylation-sensitive restrictionenzymes which cannot cleave sequences that contain methylated CpG sitesto assess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of161P2F10B. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 161P2F10B expression. The presence of RT-PCRamplifiable 161P2F10B mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting161P2F10B mRNA or 161P2F10B protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 161P2F10BmRNA expression correlates to the degree of susceptibility. In aspecific embodiment, the presence of 161P2F10B in prostate or othertissue is examined, with the presence of 161P2F10B in the sampleproviding an indication of prostate cancer susceptibility (or theemergence or existence of a prostate tumor). Similarly, one can evaluatethe integrity 161P2F10B nucleotide and amino acid sequences in abiological sample, in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike. The presence of one or more perturbations in 161P2F10B geneproducts in the sample is an indication of cancer susceptibility (or theemergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 161P2F10B mRNA or 161P2F10B proteinexpressed by tumor cells, comparing the level so determined to the levelof 161P2F10B mRNA or 161P2F10B protein expressed in a correspondingnormal tissue taken from the same individual or a normal tissuereference sample, wherein the degree of 161P2F10B mRNA or 161P2F10Bprotein expression in the tumor sample relative to the normal sampleindicates the degree of aggressiveness. In a specific embodiment,aggressiveness of a tumor is evaluated by determining the extent towhich 161P2F10B is expressed in the tumor cells, with higher expressionlevels indicating more aggressive tumors. Another embodiment is theevaluation of the integrity of 161P2F10B nucleotide and amino acidsequences in a biological sample, in order to identify perturbations inthe structure of these molecules such as insertions, deletions,substitutions and the like. The presence of one or more perturbationsindicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 161P2F10B mRNA or161P2F10B protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 161P2F10B mRNA or 161P2F10Bprotein expressed in an equivalent tissue sample taken from the sameindividual at a different time, wherein the degree of 161P2F10B mRNA or161P2F10B protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining 161P2F10Bexpression in the tumor cells over time, where increased expression overtime indicates a progression of the cancer. Also, one can evaluate theintegrity 161P2F10B nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,where the presence of one or more perturbations indicates a progressionof the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 161P2F10B gene and161P2F10B gene products (or perturbations in 161P2F10B gene and161P2F10B gene products) and a factor that is associated withmalignancy, as a means for diagnosing and prognosticating the status ofa tissue sample. A wide variety of factors associated with malignancycan be utilized, such as the expression of genes associated withmalignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.)as well as gross cytological observations (see, e.g., Bocking et al.,1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol.26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden etal., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing acoincidence between the expression of 161P2F10B gene and 161P2F10B geneproducts (or perturbations in 161P2F10B gene and 161P2F10B geneproducts) and another factor that is associated with malignancy areuseful, for example, because the presence of a set of specific factorsthat coincide with disease provides information crucial for diagnosingand prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 161P2F10B gene and 161P2F10B gene products (orperturbations in 161P2F10B gene and 161P2F10B gene products) and anotherfactor associated with malignancy entails detecting the overexpressionof 161P2F10B mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSMexpression), and observing a coincidence of 161P2F10B mRNA or proteinand PSA mRNA or protein overexpression (or PSCA or PSM expression). In aspecific embodiment, the expression of 161P2F10B and PSA mRNA inprostate tissue is examined, where the coincidence of 161P2F10B and PSAmRNA overexpression in the sample indicates the existence of prostatecancer, prostate cancer susceptibility or the emergence or status of aprostate tumor.

Methods for detecting and quantifying the expression of 161P2F10B mRNAor protein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 161P2F10B mRNAinclude in situ hybridization using labeled 161P2F10B riboprobes,Northern blot and related techniques using 161P2F10B polynucleotideprobes, RT-PCR analysis using primers specific for 161P2F10B, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR is used to detect and quantify 161P2F10B mRNAexpression. Any number of primers capable of amplifying 161P2F10B can beused for this purpose, including but not limited to the various primersets specifically described herein. In a specific embodiment, polyclonalor monoclonal antibodies specifically reactive with the wild-type161P2F10B protein can be used in an immunohistochemical assay ofbiopsied tissue.

IX.) Identification of Molecules that Interact with 161P2F10B

The 161P2F10B protein and nucleic acid sequences disclosed herein allowa skilled artisan to identify proteins, small molecules and other agentsthat interact with 161P2F10B, as well as pathways activated by 161P2F10Bvia any one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 November 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 161P2F10B protein sequences. In such methods,peptides that bind to 161P2F10B are identified by screening librariesthat encode a random or controlled collection of amino acids. Peptidesencoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the 161P2F10B protein.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 161P2F10B proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 161P2F10B are used to identifyprotein-protein interactions mediated by 161P2F10B. Such interactionscan be examined using immunoprecipitation techniques (see, e.g.,Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).161P2F10B protein can be immunoprecipitated from 161P2F10B-expressingcell lines using anti-161P2F10B antibodies. Alternatively, antibodiesagainst His-tag can be used in a cell line engineered to express fusionsof 161P2F10B and a His-tag (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as Western blotting, ³⁵S-methionine labeling ofproteins, protein microsequencing, silver staining and two-dimensionalgel electrophoresis.

Small molecules and ligands that interact with 161P2F10B can beidentified through related embodiments of such screening assays. Forexample, small molecules can be identified that interfere with proteinfunction, including molecules that interfere with 161P2F10B's ability tomediate phosphorylation and de-phosphorylation, interaction with DNA orRNA molecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 161P2F10B-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 161P2F10B (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 161P2F10B function can beidentified based on their ability to bind 161P2F10B and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of161P2F10B and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 161P2F10B.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 161P2F10B amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with the 161P2F10B amino acid sequence, allowing thepopulation of molecules and the 161P2F10B amino acid sequence tointeract under conditions that facilitate an interaction, determiningthe presence of a molecule that interacts with the 161P2F10B amino acidsequence, and then separating molecules that do not interact with the161P2F10B amino acid sequence from molecules that do. In a specificembodiment, the method further comprises purifying, characterizing andidentifying a molecule that interacts with the 161P2F10B amino acidsequence. The identified molecule can be used to modulate a functionperformed by 161P2F10B. In a preferred embodiment, the 161P2F10B aminoacid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 161P2F10B as a protein that is normally expressedin a restricted set of tissues, but which is also expressed in prostateand other cancers, opens a number of therapeutic approaches to thetreatment of such cancers. As contemplated herein, 161P2F10B functionsas a transcription factor involved in activating tumor-promoting genesor repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the161P2F10B protein are useful for patients suffering from a cancer thatexpresses 161P2F10B. These therapeutic approaches generally fall intotwo classes. One class comprises various methods for inhibiting thebinding or association of the 161P2F10B protein with its binding partneror with other proteins. Another class comprises a variety of methods forinhibiting the transcription of the 161P2F10B gene or translation of161P2F10B mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 161P2F10B-relatedprotein or 161P2F10B-related nucleic acid. In view of the expression of161P2F10B, cancer vaccines prevent and/or treat 161P2F10B-expressingcancers with minimal or no effects on non-target tissues. The use of atumor antigen in a vaccine that generates humoral and/or cell-mediatedimmune responses as anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 161P2F10B-relatedprotein, or an 161P2F10B-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 161P2F10B immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., CancerImmunol Immunother 2000 June 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in the 161P2F10B proteinshown in SEQ ID NO: 703 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, the 161P2F10B immunogen contains a biological motif,see e.g., Tables V-XVIII, or a peptide of a size range from 161P2F10Bindicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 161P2F10B protein, immunogenic regions or epitopes thereofcan be combined and delivered by various means. Such vaccinecompositions can include, for example, lipopeptides (e.g., Vitiello, A.et al., J. Clin. Invest. 95:341, 1995), peptide compositionsencapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see,e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),peptide compositions contained in immune stimulating complexes (ISCOMS)(see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., ClinExp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 161P2F10B-associated cancer, the vaccine compositionsof the invention can also be used in conjunction with other treatmentsused for cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines

CTL epitopes can be determined using specific algorithms to identifypeptides within 161P2F10B protein that bind corresponding HLA alleles(see e.g., Table IV; Epimer™ and Epimatrix™, Brown University; and,BIMAS. In a preferred embodiment, the 161P2F10B immunogen contains oneor more amino acid sequences identified using techniques well known inthe art, such as the sequences shown in Tables V-XVIII or a peptide of8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif(e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide ofat least 9 amino acids that comprises an HLA Class II motif/supermotif(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, theHLA Class I binding groove is essentially closed ended so that peptidesof only a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a peptide of about 9 or more amino acids can be bound by anHLA Class II molecule. Due to the binding groove differences between HLAClass I and II, HLA Class I motifs are length specific, i.e., positiontwo of a Class I motif is the second amino acid in an amino to carboxyldirection of the peptide. The amino acid positions in a Class II motifare relative only to each other, not the overall peptide, i.e.,additional amino acids can be attached to the amino and/or carboxyltermini of a motif-bearing sequence. HLA Class II epitopes are often 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. the 161P2F10B protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 161P2F10B in a host, by contacting the host with asufficient amount of at least one 161P2F10B B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 161P2F10B B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 161P2F10B-related protein or aman-made multiepitopic peptide comprising: administering 161P2F10Bimmunogen (e.g. the 161P2F10B protein or a peptide fragment thereof, an161P2F10B fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 161P2F10B immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 161P2F10B immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 161P2F10B, in order to generate a response tothe target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 161P2F10B. Constructscomprising DNA encoding a 161P2F10B-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 161P2F10B protein/immunogen.Alternatively, a vaccine comprises a 161P2F10B-related protein.Expression of the 161P2F10B-related protein immunogen results in thegeneration of prophylactic or therapeutic humoral and cellular immunityagainst cells that bear 161P2F10B protein. Various prophylactic andtherapeutic genetic immunization techniques known in the art can beused. Nucleic acid-based delivery is described, for instance, in Wolffet. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859;5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720.Examples of DNA-based delivery technologies include “naked DNA”,facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationiclipid complexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 161P2F10B-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 161P2F10B-relatednucleic acid molecule. In one embodiment, the full-length human161P2F10B cDNA is employed. In another embodiment, 161P2F10B nucleicacid molecules encoding specific cytotoxic T lymphocyte (CTL) and/orantibody epitopes are employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 161P2F10B antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 161P2F10B peptides to T cells in the context of MHC class I orII molecules. In one embodiment, autologous dendritic cells are pulsedwith 161P2F10B peptides capable of binding to MHC class I and/or classII molecules. In another embodiment, dendritic cells are pulsed with thecomplete 161P2F10B protein. Yet another embodiment involves engineeringthe overexpression of the 161P2F10B gene in dendritic cells usingvarious implementing vectors known in the art, such as adenovirus(Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Hendersonet al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associatedvirus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869),or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 161P2F10B can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) 161P2F10B as a Target for Antibody-Based Therapy

161P2F10B is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 161P2F10B is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of161P2F10B-immunoreactive compositions are prepared that exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunoreactive composition tonon-target organs and tissues. Antibodies specifically reactive withdomains of 161P2F10B are useful to treat 161P2F10B-expressing cancerssystemically, either as conjugates with a toxin or therapeutic agent, oras naked antibodies capable of inhibiting cell proliferation orfunction.

161P2F10B antibodies can be introduced into a patient such that theantibody binds to 161P2F10B and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 161P2F10B,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 161P2F10B sequence shown in FIG. 2 or FIG. 3.In addition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 161P2F10B), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-161P2F10B antibody) that binds to a marker (e.g.161P2F10B) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment is a method of delivering a cytotoxicand/or therapeutic agent to a cell expressing 161P2F10B, comprisingconjugating the cytotoxic agent to an antibody that immunospecificallybinds to a 161P2F10B epitope, and, exposing the cell to theantibody-agent conjugate. Another illustrative embodiment is a method oftreating an individual suspected of suffering from metastasized cancer,comprising a step of administering parenterally to said individual apharmaceutical composition comprising a therapeutically effective amountof an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-161P2F10B antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of Y⁹¹ or I¹³¹ toanti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. orBexxar™, Coulter Pharmaceuticals), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 161P2F10B antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation.

Although 161P2F10B antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 161P2F10Bexpression, preferably using immunohistochemical assessments of tumortissue, quantitative 161P2F10B imaging, or other techniques thatreliably indicate the presence and degree of 161P2F10B expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-161P2F10B monoclonal antibodies that treat prostate and othercancers include those that initiate a potent immune response against thetumor or those that are directly cytotoxic. In this regard,anti-161P2F10B monoclonal antibodies (mAbs) can elicit tumor cell lysisby either complement-mediated or antibody-dependent cell cytotoxicity(ADCC) mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-161P2F10B mAbs thatexert a direct biological effect on tumor growth are useful to treatcancers that express 161P2F10B. Mechanisms by which directly cytotoxicmAbs act include: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-161P2F10B mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 161P2F10Bantigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-161P2F10B mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-161P2F10BmAbs can be administered concomitantly with other therapeuticmodalities, including but not limited to various chemotherapeuticagents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF),surgery or radiation. The anti-161P2F10B mAbs are administered in their“naked” or unconjugated form, or can have a therapeutic agent(s)conjugated to them.

Anti-161P2F10B antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of theanti-161P2F10B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general,doses in the range of 10-1000 mg mAb per week are effective and welltolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-161P2F10B mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 161P2F10B expression in the patient, the extentof circulating shed 161P2F10B antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 161P2F10B ina given sample (e.g. the levels of circulating 161P2F10B antigen and/or161P2F10B expressing cells) in order to assist in the determination ofthe most effective dosing regimen, etc. Such evaluations are also usedfor monitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-161P2F10B antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 161P2F10B-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-161P2F10B antibodiesthat mimic an epitope on a 161P2F10B-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 161P2F10B as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 161P2F10B antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1.) Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 161P2F10B, the PADRE®universal helper T cell epitope (or multiple HTL epitopes from161P2F10B), and an endoplasmic reticulum-translocating signal sequencecan be engineered. A vaccine may also comprise epitopes that are derivedfrom other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. NatlAcad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2.) Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:760[[710]]), Plasmodium falciparum circumsporozoite (CS) protein atpositions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 761[[711]]), andStreptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQID NO: 762[[712]]). Other examples include peptides bearing a DR 1-4-7supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 763[[713]]), where “X” iseither cyclohexylalanine, phenylalanine, or tyrosine, and a is eitherD-alanine or L-alanine, has been found to bind to most HLA-DR alleles,and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type. An alternative of a pan-DRbinding epitope comprises all “L” natural amino acids and can beprovided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3.) Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4.) Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 161P2F10B. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 161P2F10B.

X.D.) Adoptive Immunotherapy

Antigenic 161P2F10B-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses161P2F10B. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 161P2F10B. Thepeptides or DNA encoding them can be administered individually or asfusions of one or more peptide sequences. Patients can be treated withthe immunogenic peptides separately or in conjunction with othertreatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 161P2F10B-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 161P2F10B, a vaccine comprising 161P2F10B-specific CTLmay be more efficacious in killing tumor cells in patient with advanceddisease than alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-161P2F10B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-161P2F10B mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 161P2F10B expression inthe patient, the extent of circulating shed 161P2F10B antigen, thedesired steady-state concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient. Non-limiting preferred human unit doses are,for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 108 to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 161P2F10B.

As disclosed herein, 161P2F10B polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

116P2F10B can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure ofthe 161P2F10B polynucleotides and polypeptides (as well as the 161P2F10Bpolynucleotide probes and anti-161P2F10B antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 161P2F10Bpolynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567 74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the161P2F10B polynucleotides described herein can be utilized in the sameway to detect 161P2F10B overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the161P2F10B polypeptides described herein can be utilized to generateantibodies for use in detecting 161P2F10B overexpression or themetastasis of prostate cells and cells of other cancers expressing thisgene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 161P2F10Bpolynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 161P2F10B-expressing cells (lymph node) is found tocontain 161P2F10B-expressing cells such as the 161P2F10B expression seenin LAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 161P2F10B polynucleotides and/or polypeptides can be usedto provide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 161P2F10B or express 161P2F10B at adifferent level are found to express 161P2F10B or have an increasedexpression of 161P2F10B (see, e.g., the 161P2F10B expression in thecancers listed in Table I and in patient samples etc. shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 161P2F10B) such as PSA, PSCA etc. (see, e.g., Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,161P2F10B polynucleotide fragments and polynucleotide variants are usedin an analogous manner. In particular, typical PSA polynucleotides usedin methods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 161P2F10Bpolynucleotide fragment is used as a probe to show the expression of161P2F10B RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 November-December 11(6):407-13 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al.eds., 1995)). Polynucleotide fragments and variants are useful in thiscontext where they are capable of binding to a target polynucleotidesequence (e.g. the 161P2F10B polynucleotide shown in SEQ ID NO: 701)under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 161P2F10B polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 161P2F10Bbiological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. the 161P2F10B polypeptide).

As shown herein, the 161P2F10B polynucleotides and polypeptides (as wellas the 161P2F10B polynucleotide probes and anti-161P2F10B antibodies orT cells used to identify the presence of these molecules) exhibitspecific properties that make them useful in diagnosing cancers such asthose listed in Table I. Diagnostic assays that measure the presence of161P2F10B gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 161P2F10Bpolynucleotides and polypeptides (as well as the 161P2F10Bpolynucleotide probes and anti-161P2F10B antibodies used to identify thepresence of these molecules) must be employed to confirm metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 161P2F10Bpolynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 161P2F10B gene maps(see Example 3 below). Moreover, in addition to their use in diagnosticassays, the 161P2F10B-related proteins and polynucleotides disclosedherein have other utilities such as their use in the forensic analysisof tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int1996 Jun. 28; 80(1-2): 63-9).

Additionally, 161P2F10B-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 161P2F10B. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to the 161P2F10B antigen. Antibodies orother molecules that react with 161P2F10B can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 161P2F10B Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 161P2F10B to its binding partner or its association withother protein(s) as well as methods for inhibiting 161P2F10B function.

XII.A.) Inhibition of 161P2F10B with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 161P2F10B are introduced into161P2F10B expressing cells via gene transfer technologies. Accordingly,the encoded single chain anti-161P2F10B antibody is expressedintracellularly, binds to 161P2F10B protein, and thereby inhibits itsfunction. Methods for engineering such intracellular single chainantibodies are well known. Such intracellular antibodies, also known as“intrabodies”, are specifically targeted to a particular compartmentwithin the cell, providing control over where the inhibitory activity ofthe treatment is focused. This technology has been successfully appliedin the art (for review, see Richardson and Marasco, 1995, TIBTECH vol.13). Intrabodies have been shown to virtually eliminate the expressionof otherwise abundant cell surface receptors (see, e.g., Richardson etal., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 161P2F10B in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 161P2F10B intrabodies inorder to achieve the desired targeting. Such 161P2F10B intrabodies aredesigned to bind specifically to a particular 161P2F10B domain. Inanother embodiment, cytosolic intrabodies that specifically bind to the161P2F10B protein are used to prevent 161P2F10B from gaining access tothe nucleus, thereby preventing it from exerting any biological activitywithin the nucleus (e.g., preventing 161P2F10B from formingtranscription complexes with other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 161P2F10B with Recombinant Proteins

In another approach, recombinant molecules bind to 161P2F10B and therebyinhibit 161P2F10B function. For example, these recombinant moleculesprevent or inhibit 161P2F10B from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 161P2F10Bspecific antibody molecule. In a particular embodiment, the 161P2F10Bbinding domain of a 161P2F10B binding partner is engineered into adimeric fusion protein, whereby the fusion protein comprises two161P2F10B ligand binding domains linked to the Fc portion of a humanIgG, such as human IgG1. Such IgG portion can contain, for example, theCH2 and CH3 domains and the hinge region, but not the CH1 domain. Suchdimeric fusion proteins are administered in soluble form to patientssuffering from a cancer associated with the expression of 161P2F10B,whereby the dimeric fusion protein specifically binds to 161P2F10B andblocks 161P2F10B interaction with a binding partner. Such dimeric fusionproteins are further combined into multimeric proteins using knownantibody linking technologies.

XII.C.) Inhibition of 161P2F10B Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 161P2F10B gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 161P2FOB mRNA into protein.

In one approach, a method of inhibiting the transcription of the161P2F10B gene comprises contacting the 161P2F10B gene with a 161P2F10Bantisense polynucleotide. In another approach, a method of inhibiting161P2F10B mRNA translation comprises contacting the 161P2F10B mRNA withan antisense polynucleotide. In another approach, a 161P2F10B specificribozyme is used to cleave the 161P2F10B message, thereby inhibitingtranslation. Such antisense and ribozyme based methods can also bedirected to the regulatory regions of the 161P2F10B gene, such as the161P2F10B promoter and/or enhancer elements. Similarly, proteins capableof inhibiting a 161P2F10B gene transcription factor are used to inhibit161P2F10B mRNA transcription. The various polynucleotides andcompositions useful in the aforementioned methods have been describedabove. The use of antisense and ribozyme molecules to inhibittranscription and translation is well known in the art.

Other factors that inhibit the transcription of 161P2F10B by interferingwith 161P2F10B transcriptional activation are also useful to treatcancers expressing 161P2F10B. Similarly, factors that interfere with161P2F10B processing are useful to treat cancers that express 161P2F10B.Cancer treatment methods utilizing such factors are also within thescope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing161P2F10B (i.e., antisense, ribozyme, polynucleotides encodingintrabodies and other 161P2F10B inhibitory molecules). A number of genetherapy approaches are known in the art. Recombinant vectors encoding161P2F10B antisense polynucleotides, ribozymes, factors capable ofinterfering with 161P2F10B transcription, and so forth, can be deliveredto target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 161P2F10B to abinding partner, etc.

In vivo, the effect of a 161P2F10B therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540, Sawyers et al., published Apr. 23, 1998, describesvarious xenograft models of human prostate cancer capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease. Efficacy can be predicted using assays that measure inhibitionof tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 161P2F10B-related protein or a 161P2F10Bgene or message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 161P2F10BGene

To isolate genes that are over-expressed in kidney cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom kidney cancer patient tissues.

The 161P2F10B SSH cDNA sequence was derived from a subtractionconsisting of a kidney cancer minus normal kidney and a mixture of 9normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney,pancreas, small intestine and heart. By RT-PCR, the 161P2F10B cDNA wasidentified as highly expressed in kidney cancer pool, with lowerexpression detected in prostate cancer xenograft pool, prostate cancerpool, colon cancer pool, lung cancer pool, ovary cancer pool, breastcancer pool, metastasis cancer pool, pancreas cancer pool, 2 differentprostate cancer metastasis to lymph node, VP1 and VP2. (FIG. 10).

The 161P2F10B SSH cDNA sequence of 182 bp matches the cDNA forphosphodiesterase I/nucleotide pyrophosphatase 3 (PDNP3). The fulllength 161P2F10B cDNA and ORF are described in FIG. 2 with the proteinsequence listed in FIG. 3.

Materials and Methods

RNA Isolation:

Tumor tissues were homogenized in Trizol reagent (Life Technologies,Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells to isolate total RNA.Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Miniand Midi kits. Total and mRNA were quantified by spectrophotometricanalysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used. DPNCDN (cDNAsynthesis primer): (SEQ ID NO:752) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQID NO:715) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ IDNO:754) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO:717)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO:756)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO:757) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO:758) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO:759) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from kidney cancer patientspecimens. The gene 161P2F10B was derived from kidney cancer patienttissues minus normal kidney and a mixture of 9 normal tissues: stomach,skeletal muscle, lung, brain, liver, kidney, pancreas, small intestineand heart. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from kidney cancer patient tissues was used as thesource of the “driver” cDNA, while the cDNA from normal tissues was usedas the source of the “tester” cDNA. Double stranded cDNAs correspondingto tester and driver cDNAs were synthesized from 2 μg of poly(A)+ RNAisolated from the relevant tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:722) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 723) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp α-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalα-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 161P2F10B gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

A typical RT-PCR expression analysis is shown in FIG. 10. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Strong expression of 161P2F10B wasobserved in kidney cancer pool. Expression was also detected in VP1,prostate cancer xenograft pool, prostate cancer pool and colon cancerpool. Low expression was observed in VP2, lung cancer pool, ovary cancerpool, breast cancer pool, metastasis pool, pancreas cancer pool, and inthe 2 different prostate cancer metastasis to lymph node.

Example 2 Full Length Cloning of 161P2F10B

To isolate genes that are involved in kidney cancer, an experiment wasconducted using kidney cancer patient specimens. The gene 161P2F10B wasderived from a subtraction consisting of kidney cancer specimens, minusnormal kidney mixed with a cocktail of 9 normal tissues: stomach,skeletal muscle, lung, brain, liver, kidney, pancreas, small intestineand heart. The SSH DNA sequence (FIG. 1) was designated 161P2F10B. cDNAclone 161P2F10B was cloned from kidney cancer specimens (FIG. 2 and FIG.3). 161P2F10B showed homology to the gene ENPP3. The amino acidalignment of 161P2F10B with ENPP3 is shown in FIG. 4 (also, see, e.g.,Buhring, et al., Blood 97:3303-3305 (2001)).

161P2F10B variant 1 was identified with a single base pair variation atnucleotide position 408 with a G instead of an A when compared to thepublished ENPP3 sequence (FIG. 2B, FIG. 3B). This nucleotide variationcoded for amino acid Lysine in variant 1, compared to amino acidarginine in ENPP3.

Example 3 Chromosomal Localization of 161P2F10B

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

161P2F10B maps to chromosome 6q22, using 161P2F10B sequence and the NCBIBLAST tool.

Example 4 Expression Analysis of 161P2F10B in Normal Tissues and PatientSpecimens

Expression of 161P2F10B was analyzed as illustrated in FIG. 10. Firststrand cDNA was prepared from vital pool 1 (VP1: liver, lung andkidney), vital pool 2 (VP2, pancreas, colon and stomach), prostatexenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancerpool, bladder cancer pool, kidney cancer pool, colon cancer pool, lungcancer pool, ovary cancer pool, breast cancer pool, metastasis cancerpool, pancreas cancer pool, and from prostate cancer metastasis to lymphnode from 2 different patients. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to161P2F10B, was performed at 26 and 30 cycles of amplification. Strongexpression of 161P2F10B was observed in kidney cancer pool. Expressionwas also detected in VP1, prostate cancer xenograft pool, prostatecancer pool and colon cancer pool. Low expression was observed in VP2,lung cancer pool, ovary cancer pool, breast cancer pool, metastasispool, pancreas cancer pool, and in the 2 different prostate cancermetastasis to lymph node.

Extensive northern blot analysis of 161P2F10B in 16 human normal tissuesconfirms the expression observed by RT-PCR (FIG. 11). Two transcripts of161P2F10B comigrating at approximately 4 kb, were detected in kidney,prostate and colon, and to lower levels, in thymus

FIG. 12 shows expression of 161P2F10B in kidney cancer xenografts. RNAwas extracted from normal kidney (N), prostate cancer xenografts,LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI, and 2 kidney cancerxenografts (Ki Xeno-1 and Ki Xeno-2). Northern blot with 10 μg of totalRNA/lane was probed with 161P2F10B sequence. The results show expressionof 161P2F10B in both kidney xenografts, LAPC-4AI, LAPC-9AI, but not innormal kidney. The expression detected in normal kidney after longexposure of the northern blot in FIG. 11, but not in FIG. 12 suggeststhat 161P2F10B is expressed at low levels in normal kidney, but isupregulated in kidney cancer.

To test expression of 161P2F10B in patient cancer specimens, RNA wasextracted from a pool of three kidney cancer patients, as well as fromnormal prostate (NP), normal bladder (NB), normal kidney (NK), normalcolon (NC). Northern blots with 10 μg of total RNA/lane were probed with161P2F10B sequence (FIG. 13). Results show expression of 161P2F10B inkidney cancer pool, but not in any of the normal tissues tested.

Analysis of individual kidney cancer tissues by northern blot showsexpression of 161P2F10B in all 4 clear cell carcinoma kidney tumors, butnot in papillary carcinoma, kidney cancer cell lines, nor in normalkidney tissues (FIG. 14).

Expression of 161P2F10B was also analyzed on kidney cancer metastasissamples (FIG. 15). RNA was extracted from kidney cancer metastasis tolung, kidney cancer metastasis to lymph node, normal bladder (NB),normal kidney (NK), and normal lung (NL), normal breast (NBr), normalovary (NO), and normal pancreas (NPa). Northern blots with 10 μg oftotal RNA/lane were probed with 161P2F10B sequence. Results show strongexpression of 161P2F10B in both kidney cancer metastasis tissues tested.Weak expression is detected in normal kidney and normal breast but notin other normal tissues.

The restricted expression of 161P2F10B in normal tissues and theupregulation detected in kidney cancer, in kidney cancer metastasis, aswell as in other cancers, suggest that 161P2F10B is a potentialtherapeutic target and a diagnostic marker for human cancers.

Example 5 Production of Recombinant 161P2F10B in Prokaryotic Systems

To express recombinant 161P2F10B in prokaryotic cells, the full orpartial length 161P2F10B cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 161P2F10B are expressed in these contructs, aminoacids 1 to 875; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidsfrom 161P2F10B, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 161P2F10B sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 161P2F10B cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 161P2F10B RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 161P2F10B at the RNA level. Transcribed 161P2F10BRNA representing the cDNA amino acid coding region of the 161P2F10B geneis used in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize161P2F10B protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 161P2F10B proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 161P2F10B cDNA protein coding sequence are fused to the GSTgene by cloning into pGEX-6P-1 or any other GST-fusion vector of thepGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). Theseconstructs allow controlled expression of recombinant 161P2F10B proteinsequences with GST fused at the amino-terminus and a six histidineepitope (6×His) at the carboxyl-terminus. The GST and 6×His tags permitpurification of the recombinant fusion protein from induced bacteriawith the appropriate affinity matrix and allow recognition of the fusionprotein with anti-GST and anti-His antibodies. The 6×His tag isgenerated by adding 6 histidine codons to the cloning primer at the 3′end, e.g., of the open reading frame (ORF). A proteolytic cleavage site,such as the PreScission™ recognition site in pGEX-6P-1, may be employedsuch that it permits cleavage of the GST tag from 161P2F10B-relatedprotein. The ampicillin resistance gene and pBR322 origin permitsselection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 161P2F10Bproteins that are fused to maltose-binding protein (MBP), all or partsof the 161P2F10B cDNA protein coding sequence are fused to the MBP geneby cloning into the pMAL-c2× and pMAL-p2× vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 161P2F10B protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 161P2F10B. The pMAL-c2× and pMAL-p2× vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 161P2F10B in bacterial cells, all or parts ofthe 161P2F10B cDNA protein coding sequence are cloned into the pETfamily of vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 161P2F10B protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 161P2F10B protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 161P2F10B in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 161P2F10B cDNA protein coding sequence are clonedinto the pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA). Thesevectors allow controlled expression from the same plasmid of up to 2different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 161P2F10B. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 161P2F10B in the yeast species Saccharomycespombe, all or parts of the 161P2F10B cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 161P2F10B protein sequence that is fusedat either the amino terminus or at the carboxyl terminus to GST whichaids purification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 6 Production of Recombinant 161P2F10B in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 161P2F10B in eukaryotic cells, the full orpartial length 161P2F10B cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 161P2F10B are expressed in these constructs, aminoacids 1 to 875; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidsfrom 161P2F10B, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-161P2F10B polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 161P2F10B in mammalian cells, the161P2F10B ORF, or portions thereof, of 161P2F10B are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 161P2F10B in mammalian cells, the161P2F10B ORF, or portions thereof, of 161P2F10B with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 161P2F10B in mammalian cellsand to allow detection of the recombinant proteins using fluorescence,the 161P2F10B ORF, or portions thereof, of 161P2F10B with a consensusKozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the GreenFluorescent Protein (GFP) fused to the carboxyl-terminus facilitatingnon-invasive, in vivo detection and cell biology studies. ThepcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene allows for selection of mammalian cells thatexpress the protein, and the ampicillin resistance gene and ColE1 originpermits selection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of the 161P2F10Bproteins.

PAPtag: The 161P2F10B ORF, or portions thereof, of 161P2F10B are clonedinto pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This constructgenerates an alkaline phosphatase fusion at the carboxyl-terminus of the161P2F10B proteins while fusing the IgGK signal sequence to theamino-terminus. Constructs are also generated in which alkalinephosphatase with an amino-terminal IgGK signal sequence is fused to theamino-terminus of 161P2F10B proteins. The resulting recombinant161P2F10B proteins are optimized for secretion into the media oftransfected mammalian cells and can be used to identify proteins such asligands or receptors that interact with the 161P2F10B proteins. Proteinexpression is driven from the CMV promoter and the recombinant proteinsalso contain myc and 6×His epitopes fused at the carboxyl-terminus thatfacilitates detection and purification. The Zeocin resistance genepresent in the vector allows for selection of mammalian cells expressingthe recombinant protein and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

ptag5: The 161P2F10B ORF, or portions thereof, of 161P2F10B are clonedinto pTag-5. This vector is similar to pAPtag but without the alkalinephosphatase fusion. This construct generates 161P2F10B protein with anamino-terminal IgGK signal sequence and myc and 6×His epitope tags atthe carboxyl-terminus that facilitate detection and affinitypurification. The resulting recombinant 161P2F10B protein is optimizedfor secretion into the media of transfected mammalian cells, and is usedas immunogen or ligand to identify proteins such as ligands or receptorsthat interact with the 161P2F10B proteins. Protein expression is drivenfrom the CMV promoter. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: The 161P2F10B ORF, or portions thereof, of 161P2F10B were clonedinto psecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, Calif.). This construct generates an IgG1 Fc fusion at theamino-terminus of the 161P2F10B proteins. 161P2F10B fusions utilizingthe murine IgG1 Fc region are also used. The resulting recombinant161P2F10B proteins are optimized for secretion into the media oftransfected mammalian cells, and can be used as immunogens or toidentify proteins such as ligands or receptors that interact with the161P2F10B protein. Protein expression is driven from the CMV promoter.The hygromycin resistance gene present in the vector allows forselection of mammalian cells that express the recombinant protein, andthe ampicillin resistance gene permits selection of the plasmid in E.coli.

-   -   pSRα Constructs: To generate mammalian cell lines that express        161P2F10B constitutively, 161P2F10B ORF, or portions thereof, of        161P2F10B are cloned into pSRα constructs. Amphotropic and        ecotropic retroviruses are generated by transfection of pSRα        constructs into the 293T-10A1 packaging line or co-transfection        of pSRα and a helper plasmid (containing deleted packaging        sequences) into the 293 cells, respectively. The retrovirus is        used to infect a variety of mammalian cell lines, resulting in        the integration of the cloned gene, 161P2F10B, into the host        cell-lines. Protein expression is driven from a long terminal        repeat (LTR). The Neomycin resistance gene present in the vector        allows for selection of mammalian cells that express the        protein, and the ampicillin resistance gene and ColE1 origin        permit selection and maintenance of the plasmid in E. coli. The        retroviral vectors can thereafter be used for infection and        generation of various cell lines using, for example, PC3, NIH        3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 161P2F10B sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ. ID. No. 764) is added tocloning primer at the 3′ end of the ORF. Additional pSRα constructs aremade to produce both amino-terminal and carboxyl-terminal GFP andmyc/6×His fusion proteins of the full-length 161P2F10B proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 161P2F10B. High virus titerleading to high level expression of 161P2F10B is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.The 161P2F10B coding sequences or fragments thereof are amplified by PCRand subcloned into the AdEasy shuttle vector (Stratagene). Recombinationand virus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 161P2F10Bcoding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 161P2F10B inmammalian cells, coding sequences of 161P2F10B, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant161P2F10B. These vectors are thereafter used to control expression of161P2F10B in various cell lines such as PC3, NIH 3T3, 293 or rat-1cells.

B. Baculovirus Expression Systems

To generate recombinant 161P2F10B proteins in a Baculovirus expressionsystem, 161P2F10B ORF, or portions thereof, are cloned into theBaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-161P2F10B isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant Baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 161P2F10B protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified Baculovirus. Recombinant161P2F10B protein can be detected using anti-161P2F10B or anti-His-tagantibody. 161P2F10B protein can be purified and used in variouscell-based assays or as immunogen to generate polyclonal and monoclonalantibodies specific for 161P2F10B.

Example 7 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 161P2F10B amino acid sequence, each assessmentavailable by accessing the ProtScale website on the ExPasy molecularbiology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 161P2F10B protein. Each of the above amino acidprofiles of 161P2F10B were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 161P2F10B protein indicated, e.g., by theprofiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 areused to prepare immunogens, either peptides or nucleic acids that encodethem, to generate therapeutic and diagnostic anti-161P2F10B antibodies.The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 161P2F10B protein. In particular, peptide immunogens ofthe invention can comprise, a peptide region of at least 5 amino acidsof FIG. 2 in any whole number increment up to 875 that includes an aminoacid position having a value greater than 0.5 in the Hydrophilicityprofile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 875 that includes an amino acidposition having a value less than 0.5 in the Hydropathicity profile ofFIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in anywhole number increment up to 875 that includes an amino acid positionhaving a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 875 that includes an amino acidposition having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8; and, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 875 that includes an aminoacid position having a value greater than 0.5 in the Beta-turn profileof FIG. 9. Peptide immunogens of the invention can also comprise nucleicacids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 161P2F10B, namely the predicted presence andlocation of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence using theHNN—Hierarchical Neural Network method (Guermeur, 1997), accessed fromthe ExPasy molecular biology server. The analysis indicates that161P2F10B is composed 31.31% alpha helix, 11.31% extended strand, and57.37% random coil (FIG. 19A).

Analysis for the potential presence of transmembrane domains in161P2F10B was carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server. Theprograms predict the presence of 1 transmembrane domain in 161P2F10B,consistent with that of a Type II cell surface protein. Showngraphically in FIG. 19 are the results of analysis using the TMpred(FIG. 19B) and TMHMM (FIG. 19C) prediction programs depicting thelocation of the transmembrane domain. The results of each program,namely the amino acids encoding the transmembrane domain are summarizedin Table XXI.

Example 8 Generation of 161P2F10B Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 161P2F10B protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 161P2F10B).

For example, 161P2F10B recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of the 161P2F10B, inwhich numerous regions are found in the predicted extracellular domaincoded by amino acids 45-870, are used as antigens to generate polyclonalantibodies in New Zealand White rabbits. For example, such regionsinclude, but are not limited to, amino acids 43-93, 100-134,211-246,467-492, 500-517, and amino acids 810-870. In addition,recombinant proteins are made that encode the whole extracellulardomain, amino acids 45-870, or halves of the domain, such as amino acids45-450 and amino acids 451-870. Antigens are also created encoding theSomatomedin-B-like domain (amino acids 53-133), the catalytic domain(amino acids 158-538), and the nuclease like domain (amino acids609-875) of 161P2F10B (Bollen et. al., 2000. Crit. Rev. Biochem. Mol.Biol., 35: 393-432), in order to generate antibodies specific to theseregions. Ideally antibodies are raised to non-conserved regions of thesedomains such that they do not crossreact with other homologousnucleotide pyrophosphatases/phosphodiesterases. It is useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude, but are not limited to, keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. In oneembodiment, a peptide encoding amino acids 500-517 of 161P2F10B isconjugated to KLH and used to immunize the rabbit. Alternatively theimmunizing agent may include all or portions of the 161P2F10B protein,analogs or fusion proteins thereof. For example, the 161P2F10B aminoacid sequence can be fused using recombinant DNA techniques to any oneof a variety of fusion protein partners that are well known in the art,such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.Such fusion proteins are purified from induced bacteria using theappropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding amino acids 45-875 isproduced and purified and used as immunogen. Other recombinant bacterialfusion proteins that may be employed include maltose binding protein,LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see thesection entitled “Production of 161P2F10B in Prokaryotic Systems” andCurrent Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M.Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M.,Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 161P2F10B in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids45-875 is cloned into the Tag5 mammalian secretion vector. Therecombinant protein is purified by metal chelate chromatography fromtissue culture supernatants of 293T cells stably expressing therecombinant vector. The purified Tag5 161P2F10B protein is then used asimmunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with Tag5 161P2F10B encoding amino acids58-538, the full-length 161P2F10B cDNA is cloned into pCDNA 3.1 myc-hisexpression vector (Invitrogen, see the Example entitled “Production ofRecombinant 161P2F10B in Eukaryotic Systems”). After transfection of theconstructs into 293T cells, cell lysates are probed with theanti-161P2F10B serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 161P2F10B protein using the Western blot technique.Immunoprecipitation and flow cytometric analyses of 293T and otherrecombinant 161P2F10B-expressing cells determine recognition of nativeprotein by the antiserum. In addition, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometrictechniques using cells that endogenously express 161P2F10B are carriedout to test specificity.

The anti-serum from the Tag5 161P2F10B immunized rabbit is affinitypurified by passage over a column composed of the Tag5 antigencovalently coupled to Affigel matrix (BioRad, Hercules, Calif.). Theserum is then further purified by protein G affinity chromatography toisolate the IgG fraction. Serum from rabbits immunized with fusionproteins, such as GST and MBP fusion proteins, are purified by depletionof antibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 9 Generation of 161P2F10B Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 161P2F10B comprise those thatreact with epitopes of the protein that would disrupt or modulate thebiological function of 161P2F10B, for example those that would disruptits catalytic activity or its interaction with ligands or proteins thatmediate its biological activity. Therapeutic mAbs also comprise thosethat specifically bind epitopes of 161P2F10B exposed on the cell surfaceand thus are useful in targeting mAb-toxin conjugates. Immunogens forgeneration of such mAbs include those designed to encode or contain theentire 161P2F10B protein, the predicted extracellular domain (aminoacids 45-875), predicted functional domains such as thesomatomedin-B-like domain (amino acids 53-133), the catalytic domain(amino acids 158-538), or the nuclease-like domain (amino acids609-875), or regions of the 161P2F10B protein predicted to be antigenicfrom computer analysis of the amino acid sequence (see, e.g., FIG. 5,FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled“Antigenicity Profiles”). Immunogens include peptides, recombinantbacterial proteins, and mammalian expressed Tag 5 proteins and human andmurine IgG FC fusion proteins. In addition, cells expressing high levelsof 161P2F10B, such as 293T-161P2F10B or 300.19-161P2F10B murine Pre-Bcells, are used to immunize mice.

To generate mAbs to 161P2F10B, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 161P2F10B-expressing cells mixed in complete Freund's adjuvant.Alternatively, mice are immunized intradermally for lymph node fusions.Mice are then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding 161P2F10B sequence is used to immunize miceby direct injection of the plasmid DNA. For example, the catalyticdomain of 161P2F10B, amino acids 158-538, is cloned into the Tag5mammalian secretion vector and the recombinant vector is used asimmunogen. In another example the same amino acids are cloned into anFc-fusion secretion vector in which the 161P2F10B sequence is fused atthe amino-terminus to an IgK leader sequence and at thecarboxyl-terminus to the coding sequence of the human or murine IgG Fcregion. This recombinant vector is then used as immunogen. The plasmidimmunization protocols are used in combination with purified proteinsexpressed from the same vector and with cells expressing 161P2F10B.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 161P2F10B monoclonal antibodies, aTag5-161P2F10B antigen encoding amino acids 158-538 is expressed andpurified from stably transfected 293T cells. Balb C mice are initiallyimmunized intraperitoneally with 25 μg of the Tag5-161P2F10B proteinmixed in complete Freund's adjuvant. Mice are subsequently immunizedevery two weeks with 25 μg of the antigen mixed in incomplete Freund'sadjuvant for a total of three immunizations. ELISA using the Tag5antigen determines the titer of serum from immunized mice. Reactivityand specificity of serum to full length 161P2F10B protein is monitoredby Western blotting, immunoprecipitation and flow cytometry using 293Tcells transfected with an expression vector encoding the 161P2F10B cDNA(see e.g., the Example entitled “Production of Recombinant 161P2F10B inEukaryotic Systems”). Other recombinant 161P2F10B-expressing cells orcells endogenously expressing 161P2F10B are also used. Mice showing thestrongest reactivity are rested and given a final injection of Tag5antigen in PBS and then sacrificed four days later. The spleens of thesacrificed mice are harvested and fused to SPO/2 myeloma cells usingstandard procedures (Harlow and Lane, 1988). Supernatants from HATselected growth wells are screened by ELISA, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometry toidentify 161P2F10B specific antibody-producing clones.

The binding affinity of a 161P2F10B monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which161P2F10B monoclonal antibodies preferred for diagnostic or therapeuticuse, as appreciated by one of skill in the art. The BIAcore system(Uppsala, Sweden) is a preferred method for determining bindingaffinity. The BIAcore system uses surface plasmon resonance (SPR,Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,Methods in Enzymology 295: 268) to monitor biomolecular interactions inreal time. BIAcore analysis conveniently generates association rateconstants, dissociation rate constants, equilibrium dissociationconstants, and affinity constants.

Example 10 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides.

Example 11 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVIIIemploy the protein sequence data from the gene product of 161P2F10B setforth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 161P2F10Bprotein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Complete protein sequences from 161P2F10B are scanned utilizing motifidentification software, to identify 8-, 9-10- and 1′-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 161P2F10B protein sequence scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of <500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 161P2F10B protein is also analyzed for the presence of 8-, 9-10-, or11-mer peptides with the HLA-B7-supermotif. Corresponding peptides aresynthesized and tested for binding to HLA-B*0702, the molecule encodedby the most common B7-supertype allele (i.e., the prototype B7 supertypeallele). Peptides binding B*0702 with IC₅₀ of <500 nM are identifiedusing standard methods. These peptides are then tested for binding toother common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, andB*5401). Peptides capable of binding to three or more of the fiveB7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the161P2F10B protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 12 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×04 CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 161P2F10B. Briefly, PBMCs are isolatedfrom patients, re-stimulated with peptide-pulsed monocytes and assayedfor the ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 13 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of500 nM or less, to three of more A2 supertype alleles. The rationale forthis requirement is that the WT peptides must be present endogenously insufficient quantity to be biologically relevant. Analoged peptides havebeen shown to have increased immunogenicity and cross-reactivity by Tcells specific for the parent epitope (see, e.g., Parkhurst et al., J.Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with161P2F10B-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 14 Identification and Confirmation of 161P2F10B-DerivedSequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 161P2F10B-derived, HLA class II HTL epitopes, the 161P2F10Bantigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 161P2F10B-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w 19,and DR9 molecules in secondary assays. Finally, peptides binding atleast two of the four secondary panel DR molecules, and thuscumulatively at least four of seven different DR molecules, are screenedfor binding to DR4w 15, DR5w11, and DR8w2 molecules in tertiary assays.Peptides binding at least seven of the ten DR molecules comprising theprimary, secondary, and tertiary screening assays are consideredcross-reactive DR binders. 161P2F10B-derived peptides found to bindcommon HLA-DR alleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 161P2F10Bantigens are analyzed for sequences carrying one of the two DR3-specificbinding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748,1994). The corresponding peptides are then synthesized and confirmed ashaving the ability to bind DR3 with an affinity of 1 μM or better, i.e.,less than 1 μM. Peptides are found that meet this binding criterion andqualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 15 Immunogenicity of 161P2F10B-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 161P2F10B-expressing tumors.

Example 16 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1-A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 17 CTL Recognition of Endogenously Processed Antigens afterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/Kb target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 161P2F10B expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 161P2F10B antigen.The choice of transgenic mouse model to be used for such an analysisdepends upon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 18 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 161P2F10B-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 161P2F10B-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100× (experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be: [(1/50,000)-(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity”. Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 19 Selection of CTL and HTL Epitopes for Inclusion in an161P2F10B-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 161P2F10B clearance. The number of epitopesused depends on observations of patients who spontaneously clear161P2F10B. For example, if it has been observed that patients whospontaneously clear 161P2F10B generate an immune response to at leastthree (3) from 161P2F10B antigen, then three or four (34) epitopesshould be included for HLA class I. A similar rationale is used todetermine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, ie., it has a high concentrationof epitopes. Epitopes may be nested or overlapping (i.e., frame shiftedrelative to one another). For example, with overlapping epitopes, two9-mer epitopes and one 10-mer epitope can be present in a 10 amino acidpeptide. Each epitope can be exposed and bound by an HLA molecule uponadministration of such a peptide. A multi-epitopic, peptide can begenerated synthetically, recombinantly, or via cleavage from the nativesource. Alternatively, an analog can be made of this native sequence,whereby one or more of the epitopes comprise substitutions that alterthe cross-reactivity and/or binding affinity properties of thepolyepitopic peptide. Such a vaccine composition is administered fortherapeutic or prophylactic purposes. This embodiment provides for thepossibility that an as yet undiscovered aspect of immune systemprocessing will apply to the native nested sequence and therebyfacilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 161P2F10B, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 161P2F10B.

Example 20 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 161P2F10B, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 161P2F10B to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HILA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-Ab-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 22 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent161P2F10B expression in persons who are at risk for tumors that bearthis antigen. For example, a polyepitopic peptide epitope composition(or a nucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 161P2F10B-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against161P2F10B-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 23 Polyepitopic Vaccine Compositions Derived from Native161P2F10B Sequences

A native 161P2F10B polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes is selected; it can be used to generate a minigene construct.The construct is engineered to express the peptide, which corresponds tothe native protein sequence. The “relatively short” peptide is generallyless than 250 amino acids in length, often less than 100 amino acids inlength, preferably less than 75 amino acids in length, and morepreferably less than 50 amino acids in length. The protein sequence ofthe vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. As noted herein, epitope motifs may be nestedor overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9-mer epitopes and one 10-merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 161P2F10B antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 161P2F10B, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 24 Polyepitopic Vaccine Compositions from Multiple Antigens

The 161P2F10B peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 161P2F10B and such other antigens.For example, a vaccine composition can be provided as a singlepolypeptide that incorporates multiple epitopes from 161P2F10B as wellas tumor-associated antigens that are often expressed with a targetcancer associated with 161P2F10B expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 25 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 161P2F10B.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 161P2F10B HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising an 161P2F10B peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 161P2F10B epitope, andthus the status of exposure to 161P2F10B, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 26 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from161P2F10B-associated disease or who have been vaccinated with an161P2F10B vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 161P2F10Bvaccine. PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50μg/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 105 irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 161P2F10B or an 161P2F10B vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 161P2F10B antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 27 Induction Of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

-   -   Group I: 3 subjects are injected with placebo and 6 subjects are        injected with 5 μg of peptide composition;    -   Group II: 3 subjects are injected with placebo and 6 subjects        are injected with 50 μg peptide composition;    -   Group III: 3 subjects are injected with placebo and 6 subjects        are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 28 Phase II Trials in Patients Expressing 161P2F10B

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses161P2F10B. The main objectives of the trial are to determine aneffective dose and regimen for inducing CTLs in cancer patients thatexpress 161P2F10B, to establish the safety of inducing a CTL and HTLresponse in these patients, and to see to what extent activation of CTLsimproves the clinical picture of these patients, as manifested, e.g., bythe reduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 161P2F10B.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 161P2F10B-associated disease.

Example 29 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 34weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against161P2F10B is generated.

Example 30 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 161P2F10B protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 161P2F10B antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 31 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 161P2F10B. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, ie., they can then betransfected with nucleic acids that encode 161P2F10B to isolate peptidescorresponding to 161P2F10B that have been presented on the cell surface.Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 32 Complementary Polynucleotides

Sequences complementary to the 161P2F10B-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 161P2F10B. Although use of oligonucleotidescomprising from about 15 to 30 base pairs is described, essentially thesame procedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 161P2F10B. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to the 161P2F10B-encodingtranscript.

Example 33 Purification of Naturally-Occurring or Recombinant 161P2F10BUsing 161P2F10B Specific Antibodies

Naturally occurring or recombinant 161P2F10B is substantially purifiedby immunoaffinity chromatography using antibodies specific for161P2F10B. An immunoaffinity column is constructed by covalentlycoupling anti-161P2F10B antibody to an activated chromatographic resin,such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing 161P2F10B are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of 161P2F10B (e.g., high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/161P2F10B binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 34 Identification of Molecules which Interact with 161P2F10B

161P2F10B, or biologically active fragments thereof, are labeled with121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem.J. 133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 161P2F10B, washed, andany wells with labeled 161P2F10B complex are assayed. Data obtainedusing different concentrations of 161P2F10B are used to calculate valuesfor the number, affinity, and association of 161P2F10B with thecandidate molecules.

Example 35 In Vivo Assay for 161P2F10B Tumor Growth Promotion

The effect of the 161P2F10B protein on tumor cell growth is evaluated invivo by gene overexpression in tumor-bearing mice. For example, SCIDmice are injected subcutaneously on each flank with 1×10⁶ of either PC3,TSUPR1, or DU145 cells containing tkNeo empty vector or 161P2F10B. Atleast two strategies may be used: (1) Constitutive 161P2F10B expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., provided suchpromoters are compatible with the host cell systems. Tumor volume isthen monitored at the appearance of palpable tumors and followed overtime and determines that 161P2F10B-expressing cells grow at a fasterrate and/or tumors produced by 161P2F10B-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine that 161P2F10B has an effect on local growthin the prostate and/or on the ability of the cells to metastasize, e.g.,to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 161P2F10B-inhibitory effect ofcandidate therapeutic compositions, such as for example, small moleculedrugs, 161P2F10B intrabodies, 161P2F10B antisense molecules andribozymes.

Example 36 161P2F10B Monoclonal Antibody-Mediated Inhibition of ProstateTumors In Vivo

The significant expression of 161P2F10B, in cancer tissues, togetherwith its restricted expression in normal tissues along with its cellsurface expression makes 161P2F10B an excellent target for antibodytherapy. Similarly, 161P2F10B is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-161P2F10B mAbs isevaluated, e.g., in human prostate cancer xenograft mouse models usingandrogen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al.,.Cancer Res, 1999. 59(19): p. 5030-6), kidney cancer xenografts (AGS-K3,AGS-K6), kidney cancer metastases to lymph node (AGS-K6 met) xenografts,and kidney cancer cell lines transfected with 161P2F10B, such as769P-161P2F10B, A498-161P2F10B.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in mouse orthotopic prostate cancer xenograft models and mousekidney xenograft models. The antibodies can be unconjugated, asdiscussed in this Example, or can be conjugated to a therapeuticmodality, as appreciated in the art. Anti-161P2F10B mAbs inhibitformation of both the androgen-dependent LAPC-9 and androgen-independentPC3-161P2F10B tumor xenografts. Anti-161P2F10B mAbs also retard thegrowth of established orthotopic tumors and prolonged survival oftumor-bearing mice. These results indicate the utility of anti-161P2F10BmAbs in the treatment of local and advanced stages of prostate cancer.(See, e.g., Saffran, D., et al., PNAS 10:1073-1078). Similarly,anti-161P2F10B mAbs can inhibit formation of AGS-K3 and AGS-K6 tumors inSCID mice, and prevent or retard the growth A498-161P2F10B tumorxenografts. These results indicate the use of anti-161P2F10B mAbs in thetreatment of prostate and/or kidney cancer.

Administration of the anti-161P2F10B mAbs leads to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 161P2F10B is anattractive target for immunotherapy and demonstrate the therapeutic useof anti-161P2F10B mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 161P2F10Bmonoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts and human kidney xenografts grown in SCIDmice.

Tumor Inhibition Using Multiple Unconjugated 161P2F10B mAbs

Materials and Methods

161P2F10B Monoclonal Antibodies:

Monoclonal antibodies are raised against 161P2F10B as described in theExample entitled “Generation of 161P2F10B Monoclonal Antibodies (mAbs)”or are obtained commercially, e.g., 97A6 (Coulter Immunotech). Theantibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 161P2F10B. Epitopemapping data for the anti-161P2F10B mAbs, as determined by ELISA andWestern analysis, recognize epitopes on the 161P2F10B protein. The 97A6antibody binds to amino acids 393-405 of the 161P2F10B protein shown inFIG. 2. Immunohistochemical analysis of cancer tissues and cells isperformed with these antibodies.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofLAPC-9 prostate tumor xenografts.

Cancer Xenografts and Cell Lines

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., supra). The AGS-K3 andAGS-K6 kidney xenografts are also passaged by subcutaneous implants in6- to 8-week old SCID mice. Single-cell suspensions of tumor cells areprepared as described in Craft, et al. The prostate carcinoma cell linePC3 (American Type Culture Collection) is maintained in RPMIsupplemented with L-glutamine and 10% FBS, and the kidney carcinoma lineA498 (American Type Culture Collection) is maintained in DMEMsupplemented with L-glutamine and 10% FBS.

PC3-161P2F10B and A498-161P2F10B cell populations are generated byretroviral gene transfer as described in Hubert, R. S., et al., STEAP: AProstate-specific Cell-surface Antigen Highly Expressed in HumanProstate Tumors, Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8.Anti-161P2F10B staining is detected by using an FITC-conjugated goatanti-mouse antibody (Southern Biotechnology Associates) followed byanalysis on a Coulter Epics-XL f low cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,AGS-K3, AGS-K6, PC3, PC3-161P2F10B, A498 or A498-161P2F10B cells mixedat a 1:1 dilution with Matrigel (Collaborative Research) in the rightflank of male SCID mice. To test antibody efficacy on tumor formation,i.p. antibody injections are started on the same day as tumor-cellinjections. As a control, mice are injected with either purified mouseIgG (ICN) or PBS; or a purified monoclonal antibody that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between mouse IgG or PBS on tumor growth. Tumorsizes are determined by vernier caliper measurements, and the tumorvolume is calculated as length×width×height. Mice with s.c. tumorsgreater than 1.5 cm in diameter are sacrificed. PSA levels aredetermined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).Circulating levels of anti-161P2F10B mAbs are determined by a captureELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran,D., et al., PNAS 10:1073-1078)

Orthotopic prostate injections are performed under anesthesia by usingketamine/xylazine. For prostate orthotopic studies, an incision is madethrough the abdominal muscles to expose the bladder and seminalvesicles, which then are delivered through the incision to expose thedorsal prostate. LAPC-9 cells (5×10⁵) mixed with Matrigel are injectedinto each dorsal lobe in a 10-μl volume. To monitor tumor growth, miceare bled on a weekly basis for determination of PSA levels. For kidneyorthopotic models, an incision is made through the abdominal muscles toexpose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel areinjected under the kidney capsule. The mice are segregated into groupsfor the appropriate treatments, with anti-161P2F10B or control mAbsbeing injected i.p.

Anti-161P2F10B mAbs Inhibit Growth of 161P2F10B-ExpressingXenograft-Cancer Tumors

The effect of anti-161P2F10B mAbs on tumor formation is tested by usingLAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c. tumormodel, the orthotopic model, which requires injection of tumor cellsdirectly in the mouse prostate or kidney, respectively, results in alocal tumor growth, development of metastasis in distal sites,deterioration of mouse health, and subsequent death (Saffran, D., etal., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90;Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make theorthotopic model more representative of human disease progression andallowed for tracking of the therapeutic effect of mAbs on clinicallyrelevant end points.

Accordingly, tumor cells are injected into the mouse prostate or kidney,and 2 days later, the mice are segregated into two groups and treatedwith either: a) 200-500 μg, of anti-161P2F10B Ab, or b) PBS three timesper week for two to five weeks.

A major advantage of the orthotopic prostate-cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a prostate-specific cell-surfaceprotein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8) oranti-G250 antibody for kidney cancer models.

Mice bearing established orthotopic LAPC-9 tumors are administered 1000μg injections of either anti-161P2F10B mAb or PBS over a 4-week period.Mice in both groups are allowed to establish a high tumor burden (PSAlevels greater than 300 ng/ml), to ensure a high frequency of metastasisformation in mouse lungs. Mice then are killed and their prostate/kidneyand lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-161P2F10Bantibodies on initiation and progression of prostate and kidney cancerin xenograft mouse models. Anti-161P2F10B antibodies inhibit tumorformation of both androgen-dependent and androgen-independent prostatetumors as well as retarding the growth of already established tumors andprolong the survival of treated mice. Moreover, anti-161P2F10B mAbsdemonstrate a dramatic inhibitory effect on the spread of local prostatetumor to distal sites, even in the presence of a large tumor burden.Similar therapeutic effects are seen in the kidney cancer model. Thus,anti-161P2F10B mAbs are efficacious on major clinically relevant endpoints (tumor growth), prolongation of survival, and health.

Example 37 Inhibition of 161P2F10B Phosphodiesterase Activity

A number of phosphodiesterase inhibitors are currently in clinicaltrials to investigate their use as vasodilators, anti-inflammatory,anti-asthmatic, anti-thrombotic and anti-depressant agents. Some ofthese inhibitors are non-selective, such as Theophylline used for thetreatment of asthma, whereas others exhibit substrate specificity. Forexample, the phosphodiesterase V inhibitor sildenafil showed greatsuccess for the treatment of male erectile dysfunction. Thephosphodiesterase I inhibitor vinpocetine is currently beinginvestigated for the treatment of urge incontinence and low compliancebladder. KF-119514 is a phosphodiesterase I/IV inhibitor shown toprotect against asthma by virtue of its anti-allergic and bronchodilatoractivities (Fujimura et al. European Journal of Pharmacology 327: 57,1997). Metformin is another phosphodiesterase I inhibitor that has beenshown to reduce lymphocyte PC-I activity after 3-months administrationto Type 2 diabetic patients, corresponding to an improvement in insulinsensitivity (Stefanovic et al. Diabetes/Metabolism Research and Reviews15:400, 1999).

The significant expression of 161P2F10B in cancer tissues, together withits restricted expression in normal tissues, makes 161P2F10B anexcellent target for phosphodiesterase inhibitor therapy. Accordingly,the efficacy of the phosphodiesterase inhibitors in human cancer mousemodels is modeled in 161P2F10B-expressing kidney, bladder or prostatecancer xenografts or cancer cell lines, either expressing 161P2F10Bendogenously, or have been engineered to express 161P2F10B as discussedin Example 6 above. Such studies performed on human pancreatic cancercells have demonstrated the utility of phosphodiesterase inhibitors inpreventing cell cycle progression of cancer cells (Boucher M et al,Biochem. Biophys. Res. Commun. 2001, 13:285). Moreover, studiesperformed in SCID mice demonstrate that phosphodiesterase inhibitorssuch as Zaprinast decrease tumor weight in neuroblastoma bearing mice(Giorgi M et al. J. Neurooncol. 2001, 51:25).

Administration of phosphodiesterase inhibitors retard established tumorgrowth and inhibit metastasis to distant sites, resulting in asignificant prolongation in the survival of tumor-bearing mice. Thesestudies show that 161P2F10B is an attractive target for cancer therapyand demonstrate the therapeutic efficacy of phosphodiesterase inhibitorsfor the treatment of local and metastatic cancers, e.g., kidney, bladderand prostate cancers.

This example demonstrates that phosphodieterase inhibitors effectivelyinhibit the growth of human tumors grown in SCID mice. Accordingly, inone embodiment it is also seen that a combination of such efficaciousphosphodiesterase inhibitors and/or derivatives thereof are alsoeffective.

Example 38 “ENPP Activity”: Detection of 161P2F10B PhosphodiesteraseActivity in Human Cancer Cells

161P2F10B is identical to ENPP3 phosphodieterase (also called CD203c orPD-1 beta). ENPP3 is an ecto-enzyme belonging to a family ofectonucleotide phosphodiesterases and pyrophospahatases. ENPP3 is aphosphodiesterase I ecto-enzyme. It is expressed in normal prostate anduterus, as well as on basophils and mast cells. Expression on thehematopoietic cells is upregulated in presence of allergen or bycross-linking with IgE (Buring et al., 1999, Blood 94: 2343).

Members of the ENPP family possess ATPase and ATP pyrophosphataseactivities. They hydrolyze extracellular nucleotides, nucleosidephosphates, and NAD. They are involved in extracellular nucleotidemetabolism, nucleotide signaling, and recycling of extracellularnucleotides. They are also involved in cell-cell and cell-matrixinteractions. ENPP enzymes differ in their substrate specificity andtissue distribution.

ENPP enzymes also play a role in recycling extracellular nucleotides. Ithas been demonstrated that ENNP1 allows activated T-cells to useNAD+from dying cells as a source of adenosine. ENPP3 expressed in theintestine may also be involved in the hydrolysis of nucleotides derivedfrom food (Byrd et al 1985, Scott et al. 1997).

To assess phosphodiesterase I activity of 161P2F10B, cell lysates orpurified protein are prepared from human normal and cancer tissues, andincubated for 5 hr at 37 degrees in 20 mM Tris/HCL, pH 9.6 containing 5mM MgCl₂ and 1 mM p-nitrophenyl thymidine-5′-L-monophosphate. Thereaction is terminated by the addition of 0.1 N NaOH and the reactionproduct quantified by reading absorbance at 410 nm (A410×64=nmolp-nitrophenol). Thus, the phosphodiesterase I activity of 161P2F10B incancer tissues is confirmed. When 161P2F10B shows phosphodiesteraseactivity, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 39 Detection of 161P2F10B Protein in Kidney Cancer PatientSpecimens

To confirm the expression of 161P2F10B protein, kidney cancer specimenswere obtained from kidney cancer patients, and stained using thecommercially available antibody 97A6 specific for ENPP3 protein (alsocalled anti-CD203c) (Immunotech, Marseilles, France). Briefly, frozentissues were cut into 4 micron sections and fixed in acetone for 10minutes. The sections were then incubated with PE-labeled mousemonoclonal anti-ENPP3 antibody for 3 hours (FIG. 16 A-F), or isotypecontrol antibody (FIG. 16G-I). The slides were washed three times inbuffer, and either analyzed by fluorescence microscopy (FIG. 16 A, B andC), or further incubated with DAKO EnVision+™ peroxidase-conjugated goatanti-mouse secondary antibody (DAKO Corporation, Carpenteria, Calif.)for 1 hour (FIG. 16 D, E, and F). The sections were then washed inbuffer, developed using the DAB kit (SIGMA Chemicals), counterstainedusing hematoxylin, and analyzed by bright field microscopy (FIG. 16 D, Eand F). The results showed strong expression of 161P2F10B in the renalcarcinoma patient tissue (FIGS. 16A and D) and the kidney cancermetastasis to lymph node tissue (FIG. 16 C and F), but weakly in normalkidney (FIG. 16 B and E). The expression was detected mostly around thecell membrane indicating that 161P2F10B is membrane associated in kidneycancer tissues. The weak expression detected in normal kidney waslocalized to the kidney tubules. The sections stained with the isotypecontrol antibody were negative showing the specificity of the anti-ENPP3antibody (FIG. 16 G-I).

FIG. 20 shows expression of 161P2F10B in human patient cancers byWestern blot analysis. Cell lysates from kidney cancer tissues (KiCa),kidney cancer metastasis to lymph node (KiCa Met), as well as normalkidney (NK) were subjected to western analysis using an anti-161P2F10Bmouse monoclonal antibody. Briefly, tissues (˜25 μg total protein) weresolubilized in SDS-PAGE sample buffer and separated on a 10-20% SDS-PAGEgel and transferred to nitrocellulose. Blots were blocked inTris-buffered saline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk. Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa. These resultsdemonstrate that 161P2F10B is useful as a diagnostic and therapeutictarget for kidney cancers, metastatic cancers and other human cancersthat express this protein.

The strong expression of 161P2F10B in kidney cancer tissues and itsrestricted expression in normal kidney as well as its membranelocalization show that 161P2F10B is a target, e.g., for kidney cancerdiagnosis and therapy. The expression detected in kidney cancermetastatic tissue indicates that 161P2F10B is also a target formetastatic disease.

Example 40 Expression of 161P2F10B Protein in Kidney Cancer XenograftTissues

Cancer xenografts were established from patient renal clear cellcarcinoma and from its metastatic tissue to lymph node. Xenografttissues were harvested from animals and dispersed into single cellsuspension. The cells were stained with the using the commerciallyavailable antibody 97A6 specific for ENPP3 protein (also calledanti-CD203c) (Immunotech, Marseilles, France). They were then washed inPBS and analyzed by flow cytometry as shown in FIG. 17. Results showstrong expression of 161P2F10B in both renal cell carcinoma xenograft(FIG. 17A) as well as renal cancer metastasis xenograft (FIG. 17B).These data demonstrate that 161P2F10B is expressed on the cell surfaceof the kidney cancer and kidney cancer metastasis xenograft cells.

FIG. 18 shows expression of 161P2F10B in xenograft tissues byimmunohistochemistry. Renal cell carcinoma (FIG. 18 A, D, G), renal cellcarcinoma metastasis to lymph node (FIG. 18 B, E, H), and prostatecancer LAPC-4AI (FIG. 18 C, F, I) xenografts were grown in SCID mice.Xenograft tissues were harvested, frozen sections were cut into 4 micronsections and fixed in acetone for 10 minutes. The sections were thenincubated with PE-labeled mouse monoclonal anti-ENPP3 antibody(Immunotech, Marseilles, France) for 3 hours (FIG. 18 A-F), or isotypecontrol antibody (FIG. 18 G-I). The slides were washed three times inbuffer, and either analyzed by fluorescence microscopy (FIG. 18 A-C), orfurther incubated with DAKO EnVision+™ peroxidase-conjugated goatanti-mouse secondary antibody (DAKO Corporation, Carpenteria, Calif.)for 1 hour (FIG. 18 D-I). The sections were then washed in buffer,developed using the DAB kit (SIGMA Chemicals), counterstained usinghematoxylin, and analyzed by bright field microscopy (FIG. 17 C-F). Theresults showed strong expression of 161P2F10B in the renal cellcarcinoma xenograft tissue (FIG. 17 A and D), in the kidney cancermetastasis to lymph node, as well as in the LAPC-4AI prostate xenograft,but not in the negative isotype control sections (FIG. 17 G, H, I). Theexpression was detected mostly around the cell membrane indicating that161P2F10B is membrane-associated in these tissues.

FIG. 21 shows expression of 161P2F10B in human xenograft tissues byWestern blot analysis. Cell lysates from kidney cancer xenograft (KiCaXeno), kidney cancer metastasis to lymph node xenograft (Met Xeno), aswell as normal kidney (NK) were subjected to Western analysis using ananti-161P2F10B mouse monoclonal antibody. Briefly, tissues (˜25 μg totalprotein) were solubilized in SDS-PAGE sample buffer and separated on a10-20% SDS-PAGE gel and transferred to nitrocellulose. Blots wereblocked in Tris-buffered saline (TBS)+3% non-fat milk and then probedwith purified anti-161P2F10B antibody in TBS+0.15% Tween-20 +1% milk.Blots were then washed and incubated with a 1:4,000 dilution ofanti-mouse IgG-HRP conjugated secondary antibody. Following washing,anti-161P2F10B immunoreactive bands were developed and visualized byenhanced chemiluminescence and exposure to autoradiographic film. Thespecific anti-161P2F10B immunoreactive bands represent a monomeric formof the 161P2F10B protein, which runs at approximately 130 kDa, and amultimer of approximately 260 kDa. These results demonstrate that thehuman cancer xenograft mouse models can be used to study the diagnosticand therapeutic effects of 161P2F10B.

The strong expression of 161P2F10B detected in kidney and prostatecancer xenograft tissues show that 161P2F10B is a cell surface target,e.g., for cancer diagnosis and therapy; these features can be modeled inhuman cancer xenograft mouse models.

Example 41 Therapeutic and Diagnostic Use of Anti-161P2F10B Antibodies(Such as Monoclonal Antibody 97A6) in Humans

Anti-161P2F10B monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. In one embodiment, the aniti-161P2F10B monoclonal antibody is97A6 (Coulter-Immunotech). The monoclonal antibody 97A6 was generatedthrough immunization of mice with erythro-megakaryoblastic cell lineUT-7 and screening for monoclonal hybridomas that specifically reactwith only a small subset of mononuclear peripheral blood cells and bonemarrow cells. Further characterization of the recognized populationdemonstrated that the mAb specifically identifies mature basophils andmast cells (Buhring, H. J. et. al. 1999. Blood 94:2343-2356). Theantigen specifically recognized by mAb 97A6 was later identified asectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) (see, e.g.,Buhring, H. J. et. al. 2001. Blood 97:3303-3305). The antibodyspecifically binds ENPP3 under native non-denatured conditions such asthose used in flow cytometric and therapeutic applications in which itis useful to bind to live cancer cells. The 97A6 mAb also specificallybinds ENPP3 under denatured/fixed conditions such as those used indiagnostic applications such as immunohistochemistry and Westernblotting.

As disclosed herein, Western blot and immunohistochemical analysis ofkidney cancer tissues and kidney cancer xenografts with mAb 97A6 showedstrong extensive staining of ENPP3 in clear cell kidney carcinoma butsignificantly lower or undetectable levels in normal kidney (FIG. 16,18, 20, 21). Detection of 161P2F10B (ENPP3) in high grade clear cellcarcinoma and in metastatic disease demonstrates the usefulness of themAb as a diagnostic and/or prognostic indicator. 97A6 is therefore usedin diagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients. ENPP3 exhibitspolarized apical surface expression in normal cells (Meerson, N. R., et.al. 2000. J. Cell Sci. 113 Pt 23:4193-4202). Detection of aredistribution of ENPP3 from apical surface expression in normal cellsto high level contiguous surface expression in advanced cancer or achange in expression levels of ENPP3 among grades of cancer by mAb 97A6also demonstrates the usefulness in diagnostic and/or prognosticapplications.

As determined by flow cytometry (FIG. 18), mAb 97A6 specifically bindsto the surface of clear cell kidney carcinoma cells. Thus,anti-161P2F10B antibodies, such as mAb 97A6 is used in diagnostic wholebody imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastatickidney cancers and other cancers that exhibit expression of 161P2F10B(ENPP3). Shedding or release of the extracellular domain of ENPP3 intothe extracellular milieu, such as that seen for alkalinephosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)),allows diagnostic detection of ENPP3 by mAb 97A6 in serum and urinesamples from suspect patients.

MAb 97A6, due to its ability to specifically bind cell surface ENPP3, isused in therapeutic applications for the treatment of cancers thatexpress ENPP3, such as clear cell kidney carcinoma. MAb 97A6 is used asan unconjugated modality and as conjugated form in which it is attachedto one of various therapeutic or imaging modalities well known in theart, such as a prodrugs, enzymes or radioisotopes. In preclinicalstudies, unconjugated and conjugated 97A6 is tested for efficacy oftumor prevention and growth inhibition in the SCID mouse kidney cancerxenograft models AGS-K3 and AGS-K6, (see, e.g., the Example entitled“Monoclonal Antibody-mediated Inhibition of Prostate and Kidney TumorsIn Vivo”. Conjugated and unconjugated 97A6 is used as a therapeuticmodality in human clinical trials either alone or in combination withother treatments as described in Examples 42-45.

Example 42 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-161P2F10B Antibodies In Vivo

Antibodies are used in accordance with the present invention, such as97A6 antibody (Coulter) which recognizes the following epitope on161P2F10B (amino acids 393-405): are used in the treatment of certainsolid tumors. Based upon a number of factors, including 161P2F10Bexpression levels among other criteria, tumors, such as those listed inTable I, e.g., breast, colon, kidney, lung, ovary, pancreas andprostate, are present preferred indications. In connection with each ofthese indications, three clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-161P2F10B antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary targets, such asthose listed above, are treated under standard protocols by the additionanti-161P2F10B antibodies to standard first and second line therapy.Protocol designs address effectiveness as assessed by reduction in tumormass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-161P2F10B antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-161P2F10Bantibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients with(cancer) tumor.

III.) Imaging Agent: Through binding a radionuclide (e.g., yttrium (90Y)) to anti-161P2F10B antibodies, the radiolabeled antibodies areutilized as a diagnostic and/or imaging agent. In such a role, thelabeled antibodies localize to both solid tumors, as well as, metastaticlesions of cells expressing 161P2F10B. In connection with the use of theanti-161P2F10B antibodies as imaging agents, the antibodies are used inassisting surgical treatment of solid tumors, as both a pre-surgicalscreen as well as a post-operative follow-up to determine what tumorremains and/or returns. In one embodiment, a (¹¹¹In)-161P2F10B antibodyis used as an imaging agent in a Phase I human clinical trial inpatients having a carcinoma that expresses 161P2F10B (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. Initial dataindicates that all primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-161P2F10B antibodies can beadministered with doses in the range of 5 to 400 mg/m 2, with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-161P2F10B antibodies relative to the affinity of a known antibodyfor its target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-161P2F10B antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-161P2F10B antibodies can be lower, perhaps in the range of 50to 300 mg/m 2, and still remain efficacious. Dosing in mg/m 2, asopposed to the conventional measurement of dose in mg/kg, is ameasurement based on surface area and is a convenient dosing measurementthat is designed to include patients of all sizes from infants toadults.

Three distinct delivery approaches are useful for delivery of theanti-161P2F10B antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allow the obtention of a high dose of the antibody atthe site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-161P2F10Bantibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-161P2F10Bantibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 161P2F10B expression levels ofpatient tumors as determined in biopsy.

As with any protein or antibody infusion based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills, (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse), and (iii) toxicity to normal cells that express 161P2F10B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-161P2F10B antibodies are found to be safe uponhuman administration.

Example 43 Human Clinical Trial Adjunctive Therapy with HumanAnti-161P2F10B Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-161P2F10B antibody in connection withthe treatment of a solid tumor, e.g., breast cancer. In the study, thesafety of single doses of anti-161P2F10B antibodies when utilized as anadjunctive therapy to an antineoplastic or chemotherapeutic agent, suchas cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, isassessed. The trial design includes delivery of six single doses of ananti-161P2F10B antibody with dosage of antibody escalating fromapproximately about 25 mg/m² to about 275 mg/m² over the course of thetreatment in accordance with the following schedule: Day 0 Day 7 Day 14Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275 mg/m ² mg/m ² mg/m ²mg/m ² mg/m ² mg/m ² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills, (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse), and (iii) toxicity to normal cells that express 161P2F10B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-161P2F10B antibodies are demonstrated to be safe andefficacious, Phase II trials confirm the efficacy and refine optimumdosing.

Example 44 Human Clinical Trial: Monotherapy with Human Anti-161P2F10BAntibody

Anti-161P2F10B antibodies are safe in connection with theabove-discussed adjunctive trial, a Phase II human clinical trialconfirms the efficacy and optimum dosing for monotherapy. Such trial isaccomplished, and entails the same safety and outcome analyses, to theabove-described adjunctive trial with the exception being that patientsdo not receive chemotherapy concurrently with the receipt of doses ofanti-161P2F10B antibodies.

Example 45 Human Clinical Trial: Diagnostic Imaging with Anti-161P2F10BAntibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-161P2F10B antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991).

Example 46 Homology Comparison of 161P2F10B to Known Sequences

The 161P2F10B gene is identical to a previously cloned and sequencedgene, namely ectonucleotide pyrophosphatase/phosphodiesterase 3 (gi4826896) (Jin-Hua P et al, Genomics 1997, 45:412), also known asphosphodiesterase-1 beta; gp130RB13-6; E-NPP3 (ENPP3), PDNP3 and CD203c.The 161P2F10B protein shows 100% identity to human ectonucleotidepyrophosphatase/phosphodiesterase 3 (gi 4826896), and 81% homology and89% identity to rat alkaline phosphodiesterase (gi 1699034). The161P2F10B protein consists of 875 amino acids, with calculated molecularweight of 100.09 kDa, and pI of 6.12. 161P2F10B is a cell surfaceprotein as shown by immunostaining in basophils (Buhring H J et al,Blood 2001, 97:3303) and in epithelial tumor cells as shown in theexample entitled “Expression of 161P2F10B protein in kidney cancerxenograft tissues”. Some localization to the golgi-endoplasmic fractionhas also been observed (Geoffroy V et al, Arch Biochem Biophys. 2001,387:154).

Two isoforms of phosphodiesterase 3 have been identified, with oneprotein containing an additional 145 aa at its amino-terminus (Choi Y Het al, Biochem J. 2001, 353:41). In addition, two variants of 161P2F10Bhave been identified. The first variant contains a single nucleotidepolymorphism (SNP) from A to G at nucleotide 408, resulting in pointmutation at amino acid 122 of the 161P2F10B protein, changing a lysineto an arginine at that position. The second variant contains a SNP (A toC) at nucleotide 2663 resulting in an amino acid change at position 383,from amino acid threonine to proline (FIG. 4C).

Motif analysis revealed the presence of several known motifs, including2-3 somatostatin B domains located at the amino terminus of the161P2F10B protein, a phosphodiesterase domain and an endonuclease domainat the C-terminus. 161P2F10B belongs to a family of closely relatedphosphodiesterases, consisting of PDNP1, -2, and -3 (Bollen M et al,Crit. Rev. Biochem Mol. Biol. 2000, 35: 393). All three members of thisfamily are type II proteins, with a short N-terminus domain locatedintracellularly. They contain one transmembrane domain, a catalyticphosphodiesterase domain and a C-terminal nuclease domain.

Phosphodiesterase 3 expression has been detected in human neoplasticsubmandibular cells, glioma cells, and tansformed lymphocytes (Murata Tet al, Anticancer Drugs 2001, 12:79; Andoh K et al, Biochim Biophys Acta1999, 1446:213; Ekholm D et al, Biochem Pharmacol 1999, 58: 935).

Phosphodiesterase 3 plays an important role in several biologicalprocesses, including release of nucleotides, cell differentiation,metabolism, cell growth, survival, angiogenesis and cell motility(Bollen M et al, Crit. Rev. Biochem Mol. Biol. 2000, 35: 393; Rawadi Get al, Endocrinol 2001, 142:4673; DeFouw L et al, Microvasc Res 2001,62:263). In addition, Phosphodiesterase 3 regulates gene expression inepithelial cells, including the expression of key adhesion moleculessuch as VCAM-1 (Blease K et al, Br J. Pharmacol. 1998, 124:229).

This information indicates that 161P2F10B plays a role in the growth ofmammalian cells, supports cell survival and motility, and regulate genetranscription by regulating events in the nucleus.

Accordingly, when 161P2F10B functions as a regulator of celltransformation, tumor formation, or as a modulator of transcriptioninvolved in activating genes associated with inflammation, tumorigenesisor proliferation, 161P2F10B is used for therapeutic, diagnostic,prognostic and/or preventative purposes. In addition, when a molecule,such as a a variant, polymorphism or SNP of 161P2F110B is expressed incancerous tissues, such as those listed in Table I, they are used fortherapeutic, diagnostic, prognostic and/or preventative purposes.

Example 47 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (J.Neurochem. 2001; 76:217-223). In particular, GPCRs have been reported toactivate MAK cascades as well as G proteins, and been associated withthe EGFR pathway in epithelial cells (Naor, Z., et al, Trends EndocrinolMetab. 2000, 11:91; Vacca F et al, Cancer Res. 2000, 60:5310; DellaRocca G J et al, J Biol. Chem. 1999, 274:13978). Usingimmunoprecipitation and Western blotting techniques, proteins areidentified that associate with 161P2F10B and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 161P2F10B, including phospholipid pathways such as P13K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as wellas mitogenic/survival cascades such as ERK, p38, etc (Cell GrowthDiffer. 2000, 11:279; J Biol. Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.).

To confirm that 161P2F10B directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

-   -   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress    -   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation    -   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress    -   4. ARE-luc, androgen receptor; steroids/MAPK;        growth/differentiation/apoptosis    -   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis    -   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 161P2F10B are mapped and used for theidentification and validation of therapeutic targets. When 161P2F10B isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 48 Involvement in Tumor Progression

The 161P2F10B gene contributes to the growth of cancer cells. The roleof 161P2F10B in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate, colon, bladder and kidneycell lines, as well as NIH 3T3 cells engineered to stably express161P2F10B. Parental cells lacking 161P2F10B and cells expressing161P2F10B are evaluated for cell growth using a well-documentedproliferation assay (Fraser, S. P., et al., Prostate, 2000; 44:61,Johnson, D. E., et al., Anticancer Drugs, 1996, 7:288).

To confirm the role of 161P2F10B in the transformation process, itseffect in colony forming assays is investigated. Parental NIH-3T3 cellslacking 161P2F10B are compared to NIH-3T3 cells expressing 161P2F10B,using a soft agar assay under stringent and more permissive conditions(Song Z. et al., Cancer Res. 2000; 60:6730).

To confirm the role of 161P2F10B in invasion and metastasis of cancercells, a well-established assay is used, e.g., a TRANSWELL Insert Systemassay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells,including prostate, colon, bladder and kidney cell lines lacking161P2F10B are compared to cells expressing 161P2F10B. Cells are loadedwith the fluorescent dye, calcein, and plated in the top well of theTRANSWELL insert coated with a basement membrane analog. Invasion isdetermined by fluorescence of cells in the lower chamber relative to thefluorescence of the entire cell population.

161P2F10B can also play a role in cell cycle and apoptosis. Parentalcells and cells expressing 161P2F10B are compared for differences incell cycle regulation using a well-established BrdU assay (Abdel-Malek,Z. A., J Cell Physiol. 1988, 136:247). In short, cells are grown underboth optimal (full serum) and limiting (low serum) conditions arelabeled with BrdU and stained with anti-BrdU Ab and propidium iodide.Cells are analyzed for entry into the G1, S, and G2M phases of the cellcycle. Alternatively, the effect of stress on apoptosis is evaluated incontrol parental cells and cells expressing 161P2F10B, including normaland tumor prostate, colon and lung cells. Engineered and parental cellsare treated with various chemotherapeutic agents, such as etoposide,flutamide, etc, and protein synthesis inhibitors, such as cycloheximide.Cells are stained with annexin V-FITC and cell death is measured by FACSanalysis. The modulation of cell death by 161P2F10B can play a criticalrole in regulating tumor progression and tumor load.

When 161P2F10B plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 49 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell, 1996, 86:353; Folkman, J.Endocrinology, 1998 139:441). Based on the effect of phsophodieseteraseinhibitors on endothelial cells, 161P2F10B plays a role in angiogenesis(DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have beendeveloped to measure angiogenesis in vitro and in vivo, such as thetissue culture assays endothelial cell tube formation and endothelialcell proliferation. Using these assays as well as in vitroneo-vascularization, the role of 161P2F10B in angiogenesis, enhancementor inhibition, is confirmed.

For example, endothelial cells engineered to express 161P2F10B areevaluated using tube formation and proliferation assays. The effect of161P2F10B is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 161P2F10B are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques.161P2F10B affects angiogenesis, and it is used as a target fordiagnostic, prognostic, preventative and/or therapeutic purposes

Example 50 Regulation of Transcription

The cell surface localization of 161P2F10B and ability to regulate VCAMexpression indicate that 161P2F10B is effectively used as a modulator ofthe transcriptional regulation of eukaryotic genes. Regulation of geneexpression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 161P2F10B. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and161P2F10B-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer,2000. 83:246). Resting cells as well as cells treated with FBS orandrogen are compared. Differentially expressed genes are identified inaccordance with procedures known in the art. The differentiallyexpressed genes are then mapped to biological pathways (Chen, K., etal., Thyroid, 2001 11:41).

In a second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, it is found that 161P2F10B plays a role in gene regulation, and itis used as a target for diagnostic, prognostic, preventative and/ortherapeutic purposes.

Example 51 Involvement in Cell Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. 161P2F10B can participate in cellular organization, and as aconsequence cell adhesion and motility. To confirm that 161P2F10Bregulates cell adhesion, control cells lacking 161P2F10B are compared tocells expressing 161P2F10B, using techniques previously described (see,e.g., Haier, et al., Br. J. Cancer, 1999, 80:1867; Lehr and Pienta, J.Natl. Cancer Inst., 1998, 90:118). Briefly, in one embodiment, cellslabeled with a fluorescent indicator, such as calcein, are incubated ontissue culture wells coated with media alone or with matrix proteins.Adherent cells are detected by fluorimetric analysis and percentadhesion is calculated. In another embodiment, cells lacking orexpressing 161P2F10B are analyzed for their ability to mediate cell-celladhesion using similar experimental techniques as described above. Bothof these experimental systems are used to identify proteins, antibodiesand/or small molecules that modulate cell adhesion to extracellularmatrix and cell-cell interaction. Cell adhesion plays a critical role intumor growth, progression, and, colonization, and 161P2F10B is involvedin these processes. Thus, 161P2F10B serves in diagnostic, prognostic,preventative and/or therapeutic modalities.

Example 52 Protein-Protein Association

Several phosophodiesterases have been shown to interact with otherproteins, thereby regulating gene transcription as well as cell growth(Butt E et al, Mol Pharmacol. 1995, 47:340). Using immunoprecipitationtechniques as well as two yeast hybrid systems, proteins are identifiedthat associate with 161P2F10B. Immunoprecipitates from cells expressing161P2F10B and cells lacking 161P2F10B are compared for specificprotein-protein associations.

Studies are performed to confirm the extent of association of 161P2F10Bwith effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phsophates etc. Studies comparing 161P2F10B positiveand 161P2F10B negative cells as well as studies comparingunstimulated/resting cells and cells treated with epithelial cellactivators, such as cytokines, growth factors, androgen andanti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr Opin Chem Biol., 1999, 3:64). A vector carryinga library of proteins fused to the activation domain of a transcriptionfactor is introduced into yeast expressing a 161P2F10B-DNA-bindingdomain fusion protein and a reporter construct. Protein-proteininteraction is detected by colorimetric reporter activity. Specificassociation with effector molecules and transcription factors directsone of skill to the mode of action of 161P2F10B, and thus identifiestherapeutic, prognostic, preventative and/or diagnostic targets forcancer. This and similar assays are also used to identify and screen forsmall molecules that interact with 161P2F10B.

Thus it is found that 161P2F10B associates with proteins and smallmolecules. Accordingly, 161P2F10 Band these proteins and small moleculesare used for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Throughout this application, various website data content, publications,patent applications and patents are referenced. The disclosures of eachof these references are hereby incorporated by reference herein in theirentireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables TABLE I Tissues that Express 161P2F10B When Malignant BreastColon Kidney Lung Ovary Pancreas Prostate

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. . A C D E F G H I K L M N P Q R S T V W Y A 40 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 C 9 −3 −4 −2 −3 −3−1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 D 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0−2 0 −1 −3 −4 −3 E 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 F 6 −3 −10 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 G 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3−2 −3 H 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 I 4 −3 2 1 −3 −3 −3 −3 −2−1 3 −3 −1 K 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 L 4 2 −3 −3 −2 −2 −2 −1 1 −2−1 M 5 −2 −2 0 −1 −1 −1 1 −1 −1 N 6 −2 0 0 1 0 −3 −4 −2 P 7 −1 −2 −1 −1−2 −4 −3 Q 5 1 0 −1 −2 −2 −1 R 5 −1 −1 −3 −3 −2 S 4 1 −2 −3 −2 T 5 0 −2−2 V 4 −3 −1 W 11 2 Y 7

TABLE IV A POSITION POSITION POSITION 2 (Primary 3 (Primary C TerminusAnchor) Anchor) (Primary Anchor) SUPERMOTIFS A1 TI LVMS FWY A2 LIVM ATQIV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYLWMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWYMIVLA MOTIFS A1TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11VTMLISAGN CDF K RYH A24 YF WM FLIW A*3101 MVT ALIS R K A*3301 MVALF ISTRK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVFWYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY

Bolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table. TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I,L A, V, I, L, P, C, S, T A, V, I, L, C, S, T,M, Y

TABLE IV C MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferredFMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWYPAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWYM W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1° anchor 1 2 31° anchor 4 5 1° anchor 6 motif a preferred LIVMFY D motif b preferredLIVMFAY DNQEST KRH DR Supermotif MFLIVWY VMSTACPLI

Italicized residues indicate less preferred or “tolerated” residues.TABLE IV D POSITION SUPER · MOTIF 1 2 3 4 5 6 7 8 C-terminus A1$\frac{1{^\circ}\quad{Anchor}}{{TI}{LVMS}}$$\frac{1{^\circ}\quad{Anchor}}{FWY}$ A2$\frac{1{^\circ}\quad{Anchor}}{{LIVM}{ATQ}}$$\frac{1{^\circ}\quad{Anchor}}{LIVMAT}$ A3 preferred$\frac{1{^\circ}\quad{Anchor}}{{VSMA}{TLI}}$ YF W(4/5) YFW (3/5) YFW(4/5) P(4/5) $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious DE(3/5); DEP(5/5) (4/5) A24 $\frac{1{^\circ}\quad{Anchor}}{{YF}{WIVLMT}}$$\frac{1{^\circ}\quad{Anchor}}{{FIY}{WLM}}$ B7 preferred FWY (5/5) LIVM(3/5) $\frac{1{^\circ}\quad{Anchor}}{P}$ FW Y(4/5) FWY (3/5)$\frac{1{^\circ}\quad{Anchor}}{{VILF}{MWYA}}$ deleterious DE(3/5); DEG(4/5) QN(4/5) DE(4/5) P(5/5); (3/5) G(4/5); A(3/5); QN(3/5) B27$\frac{1{^\circ}\quad{Anchor}}{RHK}$$\frac{1{^\circ}\quad{Anchor}}{{FYL}{WMIVA}}$ B44$\frac{1{^\circ}\quad{Anchor}}{ED}$$\frac{1{^\circ}\quad{Anchor}}{FWYLIMVA}$ B58$\frac{1{^\circ}\quad{Anchor}}{ATS}$$\frac{1{^\circ}\quad{Anchor}}{{FWY}{LIVMA}}$ B62$\frac{1{^\circ}\quad{Anchor}}{{QL}{IVMP}}$$\frac{1{^\circ}\quad{Anchor}}{{FWY}{MIVLA}}$

TABLE IV E 9 or C- C- 1 2 3 4 5 6 7 8 terminus terminus A1 9-merpreferred GFYW $\frac{1{^\circ}\quad{Anchor}}{STM}$ DEA YFW P DEQN YFW$\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious DE RHKLIVMP A G A A19-mer preferred GRHK ASTCLIV M $\frac{1{^\circ}\quad{Anchor}}{{DE}{AS}}$GSTC ASTC LIVM DE $\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious ARHKDEP DE PQN RHK PG GP YFW A1 10-mer preferred YFW$\frac{1{^\circ}\quad{Anchor}}{STM}$ DEAQN A YFW QN PASTC GDE P$\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious GP RHKGLIVM DE RHK QNARHKYF RHK A W A1 10-mer preferred YEW STCLIV M$\frac{1{^\circ}\quad{Anchor}}{{DE}{AS}}$ A YFW PG G YFW$\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious RHK RHKDEP P G PRHK QN YFW A2.1 9-mer preferred YFW $\frac{1{^\circ}\quad{Anchor}}{{LM}{IVQAT}}$YFW STC YFW A P $\frac{1{^\circ}\quad{Anchor}}{V{LIMAT}}$ deleteriousDEP DERKH RKH DERKH A2.1 10-mer preferred AYFW$\frac{1{^\circ}\quad{Anchor}}{{LM}{IVQAT}}$ LVIM G G FYWL VIM$\frac{1{^\circ}\quad{Anchor}}{V{LIMAT}}$ deleterious DEP DE RKH P RKHDERK RKH A H A3 preferred RHK$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{LMVISA} \\{TFCGD}\end{matrix}}$ YFW PRHK YFW A YFW P$\frac{1{^\circ}\quad{Anchor}}{{KYR}{HFA}}$ deleterious DEP DE A11preferred A $\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{VTLMIS} \\{{AGN}{CDF}}\end{matrix}}$ YFW YFW A YFW YFW P$\frac{1{^\circ}\quad{Anchor}}{K{RYH}}$ deleterious DEP A G A24 9-merpreferred YFWR HK $\frac{1{^\circ}\quad{Anchor}}{{YFW}M}$ STC YFW YFW$\frac{1{^\circ}\quad{Anchor}}{FLIW}$ deleterious DEG DE G QNP DERH GAQN K A24 10-mer preferred $\frac{1{^\circ}\quad{Anchor}}{{YFW}M}$ P YFWP P $\frac{1{^\circ}\quad{Anchor}}{FLIW}$ deleterious GDE QN RHK DE A QNDEA A3101 preferred RHK $\frac{1{^\circ}\quad{Anchor}}{{MVT}{ALIS}}$ YFWP YFW YFW AP $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious DEP DE ADEDE DE DE A3301 preferred $\frac{1{^\circ}\quad{Anchor}}{{MVALF}{IST}}$YFW AYFW $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious GP DE A6801preferred YFWST C $\frac{1{^\circ}\quad{Anchor}}{{AVT}{MSLI}}$ YFW LIV MYFW P $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious GP DEG RHK A B0702preferred RHKF WY $\frac{1{^\circ}\quad{Anchor}}{P}$ RHK RHK RHK RHK PA$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{{LMF}{WY}} \\{AIV}\end{matrix}}$ deleterious DEQNP DEP DE DE GDE QN DE B3501 preferredFWYLI VM $\frac{1{^\circ}\quad{Anchor}}{P}$ FWY FWY$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{LMFWY} \\{IVA}\end{matrix}}$ deleterious AGP G G B51 preferred LIVMF WY$\frac{1{^\circ}\quad{Anchor}}{P}$ FWY STC FWY G FWY$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{{LIVF}{WY}} \\{AM}\end{matrix}}$ deleterious AGPDE DE G DEQN GDE RHKST C B5301 preferredLIVMF WY $\frac{1{^\circ}\quad{Anchor}}{P}$ FWY STC FWY LIVMF WY FWY$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{IMFWY} \\{ALV}\end{matrix}}$ deleterious AGPQN G RHKQN DE B5401 preferred FWY$\frac{1{^\circ}\quad{Anchor}}{P}$ FWYLIVM LIV M ALIVM FWYA P$\frac{1{^\circ}\quad{Anchor}}{\begin{matrix}{{ATIV}{LM}} \\{FWY}\end{matrix}}$ deleterious GPQND GDESTC RHK DE QNDGE DE E DEItalicized residues indicate less preferred or “tolerated” residues. Theinformation in this Table is specific for 9-mers unless otherwisespecified.

TABLE V HLA PEPTIDE SCORING RESULTS-161P2F10B-A1, 9-MERS SCORE (ESTIMATEOF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF START RESIDUE AMOLECULE CONTANING SEQ. RANK POSITION LISTING THIS SUBSEQUENCE) ID# 1165 SMDGFRAEY 25.000 1. 2 814 NVESCPEGK 18.000 2. 3 115 CSDDCLQKK 15.0003. 4 506 KTEVEPFEN 11.250 4. 5 305 WLDLPKAER 10.000 5. 6 858 VSEILQLKT6.750 6. 7 431 KPDQHFKPY 6.250 7. 8 377 QTYCNKMEY 6.250 8. 9 374GMDQTYCNK 5.000 9. 10 70 RCDVACKDR 5.000 10. 11 514 NIEVYNLMC 4.500 11.12 382 KMEYMTDYF 4.500 12. 13 89 CVESTRIWM 4.500 13. 14 800 WLDVLPFII2.500 14. 15 462 FVDQQWLAV 2.500 15. 16 619 NVDHCLLYH 2.500 16. 17 771NTDVPIPTH 2.500 17. 18 178 DTLMPNINK 2.500 18. 19 710 ITSNLVPMY 2.50019. 20 745 VVSGPIFDY 2.500 20. 21 491 SMEAIFLAH 2.250 21. 22 8 ATEQPVKKN2.250 22. 23 670 RVPPSESQK 2.000 23. 24 439 YLTPDLPKR 2.000 24. 25 673PSESQKCSF 1.350 25. 26 273 GSEVAINGS 1.350 26. 27 419 NSEEIVRNL 1.35027. 28 188 KTCGIHSKY 1.250 28. 29 386 MTDYFPRIN 1.250 29. 30 486NNEFRSMEA 1.125 30. 31 572 SLDCFCPHL 1.000 31. 32 713 NLVPMYEEF 1.00032. 33 692 FLYPPASNR 1.000 33. 34 42 GLGLRKLEK 1.000 34. 35 114SCSDDCLQK 1.000 35. 36 126 CADYKSVCQ 1.000 36. 37 223 IIDNNMYDV 1.00037. 38 666 RADVRVPPS 1.000 38. 39 585 QLEQVNQML 0.900 39. 40 65GLENCRCDV 0.900 40. 41 827 WVEERFTAH 0.900 41. 42 139 WLEENCDTA 0.90042. 43 107 RLEASLCSC 0.900 43. 44 508 EVEPFENIE 0.900 44. 45 597QEEITATVK 0.900 45. 46 47 KLEKQGSCR 0.900 46. 47 552 HAEEVSKFS 0.900 47.48 701 TSDSQYDAL 0.750 48. 49 686 KNITHGFLY 0.625 49. 50 618 KNVDHCLLY0.625 50.

TABLE VI HLA PEPTIDE SCORING RESULTS-161P2F10B-A1, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 858 VSEILQLKTY 67.500 51. 2 386 MTDYFPRINF 62.500 52.3 771 NTDVPIPTHY 62.500 53. 4 841 DVELLTGLDF 45.000 54. 5 508 EVEPFENIEV45.000 55. 6 280 GSFPSIYMPY 37.500 56. 7 441 TPDLPKRLHY 31.250 57. 8 370LADHGMDQTY 25.000 58. 9 746 VSGPIFDYNY 15.000 59. 10 273 GSEVAINGSF13.500 60. 11 619 NVDHCLLYHR 10.000 61. 12 462 FVDQQWLAVR 10.000 62. 13673 PSESQKCSFY 6.750 63. 14 552 HAEEVSKFSV 4.500 64. 15 627 HREYVSGFGK4.500 65. 16 585 QLEQVNQMLN 4.500 66. 17 596 TQEEITATVK 2.700 67. 18 758HFDAPDEITK 2.500 68. 19 744 NVVSGPIFDY 2.500 69. 20 344 VVDHAFGMLM 2.50070. 21 506 KTEVEPFENI 2.250 71. 22 8 ATEQPVKKNT 2.250 72. 23 718YEEFRKMWDY 2.250 73. 24 822 KPEALWVEER 2.250 74. 25 802 DVLPFIIPHR 2.00075. 26 179 TLMPNINKLK 2.000 76. 27 827 WVEERFTAHI 1.800 77. 28 47KLEKQGSCRK 1.800 78. 29 701 TSDSQYDALI 1.500 79. 30 164 FSMDGFRAEY 1.50080. 31 113 CSCSDDCLQK 1.500 81. 32 419 NSEEIVRNLS 1.350 82. 33 409HNIPHDFFSF 1.250 83. 34 388 DYFPRINFFY 1.250 84. 35 155 GFDLPPVILF 1.25085. 36 661 VPDCLRADVR 1.250 86. 37 217 YPESHGIIDN 1.125 87. 38 713NLVPMYEEFR 1.000 88. 39 714 LVPMYEEFRK 1.000 89. 40 38 GLGLGLGLRK 1.00090. 41 5 LTLATEQPVK 1.000 91. 42 709 LITSNLVPMY 1.000 92. 43 89CVESTRJWMC 0.900 93. 44 139 WLEENCDTAQ 0.900 94. 45 170 RAEYLYTWDT 0.90095. 46 107 RLEASLCSCS 0.900 96. 47 491 SMEAIFLAHG 0.900 97. 48 65GLENCRCDVA 0.900 98. 49 416 FSFNSEEIVR 0.750 99. 50 572 SLDCFCPHLQ 0.500100.

TABLE VII HLA PEPTIDE SCORING RESULTS-161P2F10B-A2, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 723 KMWDYFHSV 11367.476 101. 2 623 CLLYHREYV 693.538102. 3 385 YMTDYFPRI 270.002 103. 4 867 YLPTFETTI 182.365 104. 5 179TLMPNINKL 181.794 105. 6 256 WLTAMYQGL 147.401 106. 7 519 NLMCDLLRI88.783 107. 8 173 YLYTWDTLM 73.129 108. 9 607 NLPFGRPRV 69.552 109. 10825 ALWVEERFT 68.037 110. 11 800 WLDVLPFII 45.649 111. 12 298 RISTLLKWL37.157 112. 13 31 LLVIMSLGL 36.316 113. 14 215 GLYPESHGI 33.385 114. 15572 SLDCFCPHL 32.471 115. 16 639 RMPMWSSYT 29.601 116. 17 27 VLLALLVIM29.468 117. 18 259 AMYQGLKAA 26.408 118. 19 455 RIDKVHLFV 21.039 119. 20462 FVDQQWLAV 19.036 120. 21 33 VIMSLGLGL 18.476 121. 22 584 TQLEQVNQM17.575 122. 23 807 IIPHRPTNV 16.258 123. 24 223 IIDNNMYDV 14.957 124. 25460 HLFVDQQWL 14.781 125. 26 847 GLDFYQDKV 13.632 126. 27 150 SQCPEGFDL12.562 127. 28 753 YNYDGHFDA 11.352 128. 29 343 QVVDHAFGM 10.337 129. 30360 NLHNCVNII 9.838 130. 31 40 GLGLGLRKL 9.827 131. 32 25 CIVLLALLV9.563 132. 33 615 VLQKNVDHC 9.518 133. 34 592 MLNLTQEEI 8.691 134. 35630 YVSGFGKAM 7.599 135. 36 862 LQLKTYLPT 7.129 136. 37 560 SVCGFANPL7.103 137. 38 865 KTYLPTFET 6.723 138. 39 490 RSMEAIFLA 6.563 139. 40 5LTLATEQPV 6.076 140. 41 337 RVIKALQVV 5.739 141. 42 22 KIACIVLLA 5.499142. 43 26 IVLLALLVI 5.415 143. 44 231 VNLNKNFSL 5.087 144. 45 594NLTQEEITA 4.968 145. 46 119 CLQKKDCCA 4.968 146. 47 443 DLPKRLHYA 4.713147. 48 860 EILQLKTYL 4.483 148. 49 65 GLENCRCDV 4.451 149. 50 512FEN1EVYNL 4.395 150.

TABLE VIII HLA PEPTIDE SCORING RESULTS-161P2F10B-A2, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 723 KMWDYFHSVL 2862.980 151. 2 397 YMYEGPAPRI 454.740152. 3 825 ALWVEERFTA 239.160 153. 4 162 ILFSMDGFRA 181.243 154. 5 692FLYPPASNRT 109.693 155. 6 439 YLTPDLPKRL 98.267 156. 7 222 GIIDNNMYDV90.183 157. 8 28 LLALLVIMSL 83.527 158. 9 30 ALLVIMSLGL 79.041 159. 10 4TLTLATEQPV 69.552 160. 11 369 LLADHGMDQT 58.537 161. 12 175 YTWDTLMPNI52.169 162. 13 639 RMPMWSSYTV 50.232 163. 14 806 FIIPHRPTNV 43.992 164.15 615 VLQKNVDHCL 36.316 165. 16 467 WLAVRSKSNT 34.279 166. 17 94RIWMCNKFRC 32.884 167. 18 584 TQLEQVNQML 32.857 168. 19 34 IMSLGLGLGL26.228 169. 20 660 TVPDCLRADV 24.952 170. 21 22 KIACIVLLAL 23.646 171.22 36 SLGLGLGLGL 21.362 172. 23 861 ILQLKTYLPT 19.003 173. 24 259AMYQGLKAAT 17.222 174. 25 594 NLTQEEITAT 17.140 175. 26 165 SMDGFRAEYL16.632 176. 27 81 CCWDFEDTCV 15.450 177. 28 591 QMLNLTQEEI 13.661 178.29 580 LQNSTQLEQV 13.511 179. 30 674 SESQKCSFYL 13.251 180. 31 708ALITSNLVPM 11.426 181. 32 111 SLCSCSDDCL 10.468 182. 33 447 RLHYAKNVRI10.433 183. 34 131 SVCQGETSWL 10.281 184. 35 180 LMPNINKLKT 9.149 185.36 360 NLHNCVNIIL 8.759 186. 37 855 VQPVSEILQL 8.469 187. 38 293VPFEERISTL 8.271 188. 39 204 KTFPNHYTIV 7.693 189. 40 64 RGLENCRCDV6.887 190. 41 595 LTQEEITATV 6.733 191. 42 157 DLPPVILFSM 4.970 192. 43342 LQVVDHAFGM 4.966 193. 44 460 HLFVDQQWLA 4.687 194. 45 827 WVEERFTAHI4.187 195. 46 431 KPDQHFKPYL 4.080 196. 47 284 SIYMPYNGSV 3.978 197. 48846 TGLDFYQDKV 3.375 198. 49 682 YLADKNITHG 3.233 199. 50 32 LVIMSLGLGL3.178 200.

TABLE IX HLA PEPTIDE SCORING RESULTS-161P2F10B-A3, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 227 NMYDVNLNK 300.000 651. 2 42 GLGLRKLEK 120.000652. 3 374 GMDQTYCNK 60.000 653. 4 692 FLYPPASNR 45.000 654. 5 302LLKWLDLPK 40.000 655. 6 397 YMYEGPAPR 30.000 656. 7 723 KMWDYFHSV 27.000657. 8 196 YMRAMYPTK 20.000 658. 9 496 FLAHGPSFK 20.000 659. 10 6TLATEQPVK 20.000 660. 11 165 SMDGFRAEY 18.000 661. 12 180 LMPNINKLK15.000 662. 13 215 GLYPESHGI 13.500 663. 14 47 KLEKQGSCR 12.000 664. 15803 VLPFIIPHR 9.000 665. 16 863 QLKTYLPTF 9.000 666. 17 439 YLTPDLPKR9.000 667. 18 351 MLMEGLKQR 6.750 668. 19 382 KMEYMTDYF 6.000 669. 20732 LLIKHATER 6.000 670. 21 305 WLDLPKAER 6.000 671. 22 162 ILFSMDGFR6.000 672. 23 38 GLGLGLGLR 5.400 673. 24 385 YMTDYFPRI 5.400 674. 25 92STRIWMCNK 4.500 675. 26 713 NLVPMYEEF 4.500 676. 27 745 VVSGPIFDY 4.050677. 28 447 RLHYAKNVR 4.000 678. 29 173 YLYTWDTLM 3.000 679. 30 341ALQVVDHAF 3.000 680. 31 670 RVPPSESQK 3.000 681. 35 843 ELLTGLDFY 2.700685. 36 179 TLMPNINKL 2.025 686. 37 204 KTFPNHYTI 2.025 687. 38 377QTYCNKMEY 2.000 688. 39 423 IVRNLSCRK 2.000 689. 40 814 NVESCPEGK 2.000690. 41 867 YLPTFETTI 1.800 691. 42 410 NIPHDFFSF 1.800 692. 43 491SMEAIFLAH 1.800 693. 44 263 GLKAATYFW 1.800 694. 45 847 GLDFYQDKV 1.800695. 46 31 LLVIMSLGL 1.800 696. 47 360 NLHNCVNII 1.800 697. 48 572SLDCFCPHL 1.800 698. 49 800 WLDVLPFII 1.800 699. 50 782 VVLTSCKNK 1.500700.

TABLE X HLA PEPTIDE SCORING RESULTS-161P2F10B-A3, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 38 GLGLGLGLRK 120.000 201. 2 186 KLKTCGIHSK 90.000202. 3 47 KLEKQGSCRK 60.000 203. 4 301 TLLKWLDLPK 60.000 204. 5 179TLMPNINKLK 33.750 205. 6 713 NLVPMYEEFR 27.000 206. 7 723 KMWDYFHSVL27.000 207. 8 6 TLATEQPVKK 20.000 208. 9 443 DLPKRLHYAK 18.000 209. 10256 WLTAMYQGLK 18.000 210. 11 350 GMLMEGLKQR 13.500 211. 12 437KPYLTPDLPK 9.000 212. 13 397 YMYEGPAPRI 6.750 213. 14 731 VLLIKHATER6.000 214. 15 714 LVPMYEEFRK 6.000 215. 16 215 GLYPESHGII 4.050 216. 17744 NVVSGPIFDY 4.050 217. 18 637 AMRMPMWSSY 4.000 218. 19 845 LTGLDFYQDK3.000 219. 20 460 HLFVDQQWLA 3.000 220. 21 825 ALWVEERFTA 3.000 221. 22162 ILFSMDGFRA 3.000 222. 23 28 LLALLVIMSL 2.700 223. 24 538 SLNHLLKVPF2.000 224. 25 286 YMPYNGSVPF 2.000 225. 26 374 GMDQTYCNKM 1.800 226. 27462 FVDQQWLAVR 1.800 227. 28 30 ALLVIMSLGL 1.800 228. 29 360 NLHNCVNIIL1.800 229. 30 619 NVDHCLLYHR 1.800 230. 31 277 AINGSFPSIY 1.800 231. 32781 FVVLTSCKNK 1.500 232. 33 5 LTLATEQPVK 1.500 233. 34 280 GSFPSIYMPY1.350 234. 35 34 IMSLGLGLGL 1.200 235. 36 603 TVKVNLPFGR 1.200 236. 37709 LITSNLVPMY 1.200 237. 38 36 SLGLGLGLGL 1.200 238. 39 822 KPEALWVEER1.080 239. 40 390 FPRINFFYMY 1.080 240. 41 494 AIFLAHGPSF 1.000 241. 4217 TLKKYKIACI 0.900 242. 43 615 VLQKNVDHCL 0.900 243. 44 165 SMDGFRAEYL0.900 244. 45 591 QMLNLTQEEI 0.900 245. 46 355 GLKQRNLHNC 0.900 246. 47422 EIVRNLSCRK 0.900 247. 48 596 TQEEITATVK 0.900 248. 49 22 KIACIVLLAL0.810 249. 50 692 FLYPPASNRT 0.750 250.

TABLE XI HLA PEPTIDE SCORING RESULTS-161P2F10B-A11, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 670 RVPPSESQK 6.000 251. 2 42 GLGLRKLEK 2.400 252. 3423 IVRNLSCRK 2.000 253. 4 814 NVESCPEGK 2.000 254. 5 227 NMYDVNLNK1.600 255. 6 782 VVLTSCKNK 1.500 256. 7 715 VPMYEEFRK 1.200 257. 8 374GMDQTYCNK 1.200 258. 9 628 REYVSGFGK 1.080 259. 10 92 STRIWMCNK 1.000260. 11 257 LTAMYQGLK 1.000 261. 12 332 GPVSARVIK 0.900 262. 13 178DTLMPNINK 0.900 263. 14 302 LLKWLDLPK 0.800 264. 15 678 KCSFYLADK 0.600265. 16 444 LPKRLHYAK 0.400 266. 17 727 YFHSVLLIK 0.400 267. 18 114SCSDDCLQK 0.400 268. 19 12 PVKKNTLKK 0.400 269. 20 496 FLAHGPSFK 0.400270. 21 714 LVPMYEEFR 0.400 271. 22 6 TLATEQPVK 0.400 272. 23 450YAKNVRIDK 0.400 273. 24 196 YMRAMYPTK 0.400 274. 25 780 YFVVLTSCK 0.300275. 26 11 QPVKKNTLK 0.300 276. 27 94 RIWMCNKFR 0.240 277. 28 38GLGLGLGLR 0.240 278. 29 447 RLHYAKNVR 0.240 279. 30 47 KLEKQGSCR 0.240280. 31 7 LATEQPVKK 0.200 281. 32 857 PVSEILQLK 0.200 282. 33 68NCRCDVACK 0.200 283. 34 180 LMPNINKLK 0.200 284. 35 162 ILFSMDGFR 0.160285. 36 397 YMYEGPAPR 0.160 286. 37 692 FLYPPASNR 0.160 287. 38 384EYMTDYFPR 0.144 288. 39 438 PYLTPDLPK 0.120 289. 40 465 QQWLAVRSK 0.120290. 41 204 KTFPNHYTI 0.120 291. 42 732 LLIKHATER 0.120 292. 43 343QVVDHAFGM 0.090 293. 44 337 RVIKALQVV 0.090 294. 45 614 RVLQKNVDH 0.090295. 46 803 VLPFIIPHR 0.080 296. 47 439 YLTPDLPKR 0.080 297. 48 351MLMEGLKQR 0.080 298. 49 417 SFNSEEIVR 0.080 299. 50 305 WLDLPKAER 0.080300.

TABLE XII HLA PEPTIDE SCORING RESULTS-161P2F10B-A11, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 714 LVPMYEEFRK 6.000 301. 2 437 KPYLTPDLPK 2.400 302.3 195 KYMRAMYPTK 2.400 303. 4 38 GLGLGLGLRK 2.400 304. 5 781 FVVLTSCKNK1.500 305. 6 5 LTLATEQPVK 1.500 306. 7 301 TLLKWLDLPK 1.200 307. 8 47KLEKQGSCRK 1.200 308. 9 186 KLKTCGIHSK 1.200 309. 10 603 TVKVNLPFGR1.200 310. 11 845 LTGLDFYQDK 1.000 311. 12 779 HYFVVLTSCK 0.800 312. 13619 NVDHCLLYHR 0.800 313. 14 449 HYAKNVRIDK 0.800 314. 15 11 QPVKKNTLKK0.600 315. 16 596 TQEEITATVK 0.600 316. 17 726 DYFHSVLLIK 0.480 317. 18462 FVDQQWLAVR 0.400 318. 19 6 TLATEQPVKK 0.400 319. 20 758 HFDAPDEITK0.400 320. 21 256 WLTAMYQGLK 0.400 321. 22 179 TLMPNINKLK 0.400 322. 23630 YVSGFGKAMR 0.400 323. 24 856 QPVSEILQLK 0.300 324. 25 495 IFLAHGPSFK0.300 325. 26 443 DLPKRLHYAK 0.240 326. 27 114 SCSDDCLQKK 0.200 327. 28428 SCRKPDQHFK 0.200 328. 29 348 AFGMLMEGLK 0.200 329. 30 691 GFLYPPASNR0.180 330. 31 802 DVLPFILPHR 0.180 331. 32 350 GMLMEGLKQR 0.180 332. 33422 EIVRNLSCRK 0.180 333. 34 10 EQPVKKNTLK 0.180 334. 35 396 FYMYEGPAPR0.160 335. 36 226 NNMYDVNLNK 0.160 336. 37 517 VYNLMCDLLR 0.160 337. 38161 VILFSMDGFR 0.120 338. 39 822 KPEALWVEER 0.120 339. 40 605 KVNLPFGRPR0.120 340. 41 713 NLVPMYEEFR 0.120 341. 42 295 FEERISTLLK 0.120 342. 43731 VLLIKHATER 0.120 343. 44 497 LAHGPSFKEK 0.100 344. 45 744 NVVSGPIFDY0.090 345. 46 189 TCGIHSKYMR 0.080 346. 47 383 MEYMTDYFPR 0.072 347. 4841 LGLGLRKLEK 0.060 348. 49 627 HREYVSGFGK 0.060 349. 50 813 TNVESCPEGK0.060 350.

TABLE XIII HLA PEPTIDE SCORING RESULTS-161P2F10B-A24, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTANING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 20 KYKIACIVL 400.000 351. 2 172 EYLYTWDTL 300.000352. 3 517 VYNLMCDLL 300.000 353. 4 388 DYFPRINFF 144.000 354. 5 216LYPESHGII 90.000 355. 6 398 MYEGPAPRI 75.000 356. 7 726 DYFHSVLLI 50.000357. 8 484 GYNNEFRSM 45.000 358. 9 100 KFRCGETRL 40.000 359. 10 378TYCNKMEYM 25.000 360. 11 58 CFDASFRGL 24.000 361. 12 348 AFGMLMEGL24.000 362. 13 854 KVQPVSEIL 20.160 363. 14 155 GFDLPPVIL 20.000 364. 15195 KYMRAMYPT 15.000 365. 16 495 IFLAHGPSF 15.000 366. 17 866 TYLPTFEIT10.800 367. 18 693 LYPPASNRT 10.800 368. 19 850 FYQDKVQPV 10.800 369. 20585 QLEQVNQML 10.080 370. 21 419 NSEEIVRNL 10.080 371. 22 720 EFRKMWDYF10.000 372. 23 488 EFRSMEAIF 10.000 373. 24 629 EYVSGFGKA 9.900 374. 25298 RISTLLKWL 9.600 375. 26 179 TLMPNINKL 9.504 376. 27 260 MYQGLKAAT9.000 377. 28 681 FYLADKNIT 9.000 378. 29 285 IYMPYNGSV 9.000 379. 30717 MYEEFRKMW 9.000 380. 31 440 LTPDLPKRL 8.640 381. 32 657 LPPTVPDCL8.400 382. 33 29 LALLVIMSL 8.400 383. 34 200 MYPTKTFPN 7.500 384. 35 24ACIVLLALL 7.200 385. 36 10 EQPVKKNTL 7.200 386. 37 37 LGLGLGLGL 7.200387. 38 860 EILQLKTYL 7.200 388. 39 33 VIMSLGLGL 7.200 389. 40 35MSLGLGLGL 7.200 390. 41 209 HYTIVTGLY 7.000 391. 42 779 HYFVVLTSC 7.000392. 43 761 APDEITKHL 6.720 393. 44 228 MYDVNLNKN 6.600 394. 45 300STLLKWLDL 6.000 395. 46 856 QPVSEILQL 6.000 396. 47 132 VCQGETSWL 6.000397. 48 587 EQVNQMLNL 6.000 398. 49 793 TPENCPGWL 6.000 399. 50 382KMEYMTDYF 6.000 400.

TABLE XIV HLA PEPTIDE SCORING RESULTS-161P2F10B-A24, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTAINING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 20 KYKIACIVLL 400.000 401. 2 705 QYDALITSNL 280.000402. 3 228 MYDVNLNKNF 120.000 403. 4 625 LYHREYVSGF 100.000 404. 5 384EYMTDYFPRI 90.000 405. 6 866 TYLPTFETTI 90.000 406. 7 629 EYVSGFGKAM37.500 407. 8 172 EYLYTWDTLM 37.500 408. 9 435 HFKPYLTPDL 28.800 409. 10488 EFRSMEAIFL 20.000 410. 11 850 FYQDKVQPVS 12.600 411. 12 584TQLEQVNQML 12.096 412. 13 452 KNVRIDKVHL 12.000 413. 14 57 KCFDASFRGL11.520 414. 15 22 KIACIVLLAL 11.200 415. 16 760 DAPDEITKHL 10.080 416.17 155 GFDLPPVILF 10.000 417. 18 750 IFDYNYDGHF 10.000 418. 19 547FYEPSHAEEV 9.900 419. 20 700 RTSDSQYDAL 9.600 420. 21 723 KMWDYFHSVL9.600 421. 22 693 LYPPASNRTS 9.000 422. 23 343 QVVDHAFGML 8.640 423. 24388 DYFPRINFFY 8.400 424. 25 615 VLQKNVDHCL 8.400 425. 26 340 KALQVVDHAF8.400 426. 27 431 KPDQHFKPYL 8.000 427. 28 178 DTLMPNINKL 7.920 428. 29752 DYNYDGHFDA 7.500 429. 30 260 MYQGLKAATY 7.500 430. 31 817 SCPEGKPEAL7.200 431. 32 103 CGETRLEASL 7.200 432. 33 571 ESLDCFCPHL 7.200 433. 3432 LVIMSLGLGL 7.200 434. 35 792 HTPENCPGWL 7.200 435. 36 559 FSVCGFANPL7.200 436. 37 255 MWLTAMYQGL 7.200 437. 38 564 FANPLPTESL 7.200 438. 39418 FNSEEIVRNL 6.720 439. 40 39 LGLGLGLRKL 6.600 440. 41 855 VQPVSEILQL6.000 441. 42 149 QSQCPEGFDL 6.000 442. 43 30 ALLVIMSLGL 6.000 443. 44352 LMEGLKQRNL 6.000 444. 45 530 APNNGTHGSL 6.000 445. 46 230 DVNLNKNFSL6.000 446. 47 607 NLPFGRPRVL 6.000 447. 48 269 YFWPGSEVAI 6.000 448. 49648 VPQLGDTSPL 6.000 449. 50 439 YLTPDLPKRL 5.760 450.

TABLE XV HLA PEPTIDE SCORING RESULTS-161P2F10B-B7, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTAINING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 403 APRIRAHNI 240.000 451. 2 390 FPRINFFYM 200.000452. 3 453 NVRIDKVHL 200.000 453. 4 836 IARVRDVEL 120.000 454. 5 856QPVSEILQL 80.000 455. 6 608 LPFGRPRVL 80.000 456. 7 776 IPTHYFVVL 80.000457. 8 657 LPPTVPDCL 80.000 458. 9 761 APDEITKHL 72.000 459. 10 612RPRVLQKNV 40.000 460. 11 818 CPEGKPEAL 24.000 461. 12 793 TPENCPGWL24.000 462. 13 516 EVYNLMCDL 20.000 463. 14 854 KVQPVSEIL 20.000 464. 15158 LPPVILFSM 20.000 465. 16 560 SVCGFANPL 20.000 466. 17 565 ANPLPTESL18.000 467. 18 29 LALLVIMSL 12.000 468. 19 24 ACIVLLALL 12.000 469. 20179 TLMPNINKL 12.000 470. 21 23 IACIVLLAL 12.000 471. 22 33 VIMSLGLGL12.000 472. 23 640 MPMWSSYTV 12.000 473. 24 344 VVDHAFGML 6.000 474. 25838 RVRDVELLT 5.000 475. 26 630 YVSGFGKAM 5.000 476. 27 343 QVVDHAFGM5.000 477. 28 534 GTHGSLNHL 4.000 478. 29 362 HNCVNIILL 4.000 479. 30 31LLVIMSLGL 4.000 480. 31 313 RPRFYTMYF 4.000 481. 32 225 DNNMYDVNL 4.000482. 33 587 EQVNQMLNL 4.000 483. 34 35 MSLGLGLGL 4.000 484. 35 440LTPDLPKRL 4.000 485. 36 300 STLLKWLDL 4.000 486. 37 643 WSSYTVPQL 4.000487. 38 675 ESQKCSFYL 4.000 488. 39 334 VSARVIKAL 4.000 489. 40 298RISTLLKWL 4.000 490. 41 774 VPIPTHYFV 4.000 491. 42 460 HLFVDQQWL 4.000492. 43 100 KFRCGETRL 4.000 493. 44 796 NCPGWLDVL 4.000 494. 45 860EILQLKTYL 4.000 495. 46 256 WLTAMYQGL 4.000 496. 47 150 SQCPEGFDL 4.000497. 48 10 EQPVKKNTL 4.000 498. 49 40 GLGLGLRKL 4.000 499. 50 37LGLGLGLGL 4.000 500.

TABLE XVI HLA PEPTIDE SCORING RESULTS-161P2F10B-B7, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTAINING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 530 APNNGTHGSL 240.000 501. 2 836 IARVRDVELL 120.000502. 3 577 CPHLQNSTQL 80.000 503. 4 648 VPQLGDTSPL 80.000 504. 5 293VPFEERISTL 80.000 505. 6 715 VPMYEEFRKM 60.000 506. 7 431 KPDQHFKPYL24.000 507. 8 343 QVVDHAFGML 20.000 508. 9 516 EVYNLMCDLL 20.000 509. 1032 LVIMSLGLGL 20.000 510. 11 230 DVNLNKNFSL 20.000 511. 12 246NPAWWHGQPM 20.000 512. 13 131 SVCQGETSWL 20.000 513. 14 564 FANPLPTESL18.000 514. 15 469 AVRSKSNTNC 15.000 515. 16 30 ALLVIMSLGL 12.000 516.17 760 DAPDELTKHL 12.000 517. 18 347 HAFGMLMEGL 12.000 518. 19 23IACIVLLALL 12.000 519. 20 403 APRIRAHNIP 6.000 520. 21 335 SARVIKALQV6.000 521. 22 154 EGFDLPPVIL 6.000 522. 23 26 IVLLALLVIM 5.000 523. 24488 EFRSMEAIFL 4.000 524. 25 584 TQLEQVNQML 4.000 525. 26 452 KNVRIDKVHL4.000 526. 27 111 SLCSCSDDCL 4.000 527. 28 599 EITATVKVNL 4.000 528. 2957 KCFDASFRGL 4.000 529. 30 418 FNSEEIVRNL 4.000 530. 31 795 ENCPGWLDVL4.000 531. 32 149 QSQCPEGFDL 4.000 532. 33 616 LQKNVDHCLL 4.000 533. 34615 VLQKNVDHCL 4.000 534. 35 700 RTSDSQYDAL 4.000 535. 36 39 LGLGLGLRKL4.000 536. 37 559 FSVCGFANPL 4.000 537. 38 36 SLGLGLGLGL 4.000 538. 3928 LLALLVIMSL 4.000 539. 40 835 HIARVRDVEL 4.000 540. 41 22 KIACIVLLAL4.000 541. 42 607 NLPFGRPRVL 4.000 542. 43 299 ISTLLKWLDL 4.000 543. 44792 HTPENCPGWL 4.000 544. 45 723 KMWDYFHSVL 4.000 545. 46 774 VPIPTHYFVV4.000 546. 47 533 NGTHGSLNHL 4.000 547. 48 390 FPRINFFYMY 4.000 548. 49817 SCPEGKPEAL 4.000 549. 50 34 IMSLGLGLGL 4.000 550.

TABLE XVII HLA PEPTIDE SCORING RESULTS-161P2F10B-B35, 9-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTAINING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 390 FPRINFFYM 120.000 551. 2 313 RPRFYTMYF 120.000552. 3 308 LPKAERPRF 90.000 553. 4 510 EPFENIEVY 80.000 554. 5 568LPTESLDCF 40.000 555. 6 158 LPPVILFSM 40.000 556. 7 253 QPMWLTAMY 40.000557. 8 856 QPVSEILQL 30.000 558. 9 193 HSKYMRAMY 30.000 559. 10 431KPDQHFKPY 24.000 560. 11 403 APRIRAHNI 24.000 561. 12 612 RPRVLQKNV24.000 562. 13 657 LPPTVPDCL 20.000 563. 14 608 LPFGRPRVL 20.000 564. 15287 MPYNGSVPF 20.000 565. 16 776 IPTHYFVVL 20.000 566. 17 556 VSKFSVCGF15.000 567. 18 761 APDEITKHL 12.000 568. 19 836 IARVRDVEL 9.000 569. 20618 KNVDHCLLY 8.000 570. 21 634 FGKAMRMPM 6.000 571. 22 793 TPENCPGWL6.000 572. 23 198 RAMYPTKTF 6.000 573. 24 818 CPEGKPEAL 6.000 574. 25293 VPFEERIST 6.000 575. 26 698 SNRTSDSQY 6.000 576. 27 407 RAHNIPHDF6.000 577. 28 35 MSLGLGLGL 5.000 578. 29 675 ESQKCSFYL 5.000 579. 30 643WSSYTVPQL 5.000 580. 31 334 VSARVIKAL 5.000 581. 32 453 NVRIDKVHL 4.500582. 33 188 KTCGIHSKY 4.000 583. 34 584 TQLEQVNQM 4.000 584. 35 343QVVDHAFGM 4.000 585. 36 640 MPMWSSYTV 4.000 586. 37 513 ENIEVYNLM 4.000587. 38 774 VPIPTHYFV 4.000 588. 39 411 IPHDFFSFN 4.000 589. 40 686KNITHUFLY 4.000 590. 41 23 IACIVLLAL 3.000 591. 42 616 LQKNVDHCL 3.000592. 43 240 SSKEQNNPA 3.000 593. 44 29 LALLVIMSL 3.000 594. 45 863QLKTYLPTF 3.000 595. 46 428 SCRKPDQHF 3.000 596. 47 545 VPFYEPSHA 3.000597. 48 671 VPPSESQKC 3.000 598. 49 221 HGIIDNNMY 3.000 599. 50 824EALWVEERF 3.000 600.

TABLE XVII HLA PEPTIDE SCORING RESULTS - 161P2F10B - B35, 9-MERSSUBSEQUENCE SCORE (ESTIMATE OF HALF TIME START RESIDUE OF DISASSOCIATIONOF A MOLECULE SEQ. RANK POSITION LISTING CONTAINING THIS SUBSEQUENCE)ID# 28 35 MSLGLGLGL 5.000 578. 29 675 ESQKCSFYL 5.000 579. 30 643WSSYTVPQL 5.000 580. 31 334 VSARVIKAL 5.000 581. 32 453 NVRIDKVHL 4.500582. 33 188 KTCGIHSKY 4.000 583. 34 584 TQLEQVNQM 4.000 584. 35 343QVVDHAFGM 4.000 585. 36 640 MPMWSSYTV 4.000 586. 37 513 ENIEVYNLM 4.000587. 38 774 VPIPTHYFV 4.000 588. 39 411 IPHDFFSFN 4.000 589. 40 686KNITHUFLY 4.000 590. 41 23 IACIVLLAL 3.000 591. 42 616 LQKNVDHCL 3.000592. 43 240 SSKEQNNPA 3.000 593. 44 29 LALLVIMSL 3.000 594. 45 863QLKTYLPTF 3.000 595. 46 428 SCRKPDQHF 3.000 596. 47 545 VPFYEPSHA 3.000597. 48 671 VPPSESQKC 3.000 598. 49 221 HGIIDNNMY 3.000 599. 50 824EALWVEERF 3.000 600.

TABLE XVIII HLA PEPTIDE SCORING RESULTS-161P2F10B-B35, 10-MERS SCORE(ESTIMATE OF SUB- HALF TIME OF DIS- SEQUENCE ASSOCIATION OF STARTRESIDUE A MOLECULE CONTAINING SEQ. RANK POSITION LISTING THISSUBSEQUENCE) ID# 1 308 LPKAERPRFY 120.000 601. 2 390 FPRINFFYMY 120.000602. 3 715 VPMYEEFRKM 60.000 603. 4 246 NPAWWHGQPM 40.000 604. 5 201YPTKTFPNHY 40.000 605. 6 293 VPFEERISTL 40.000 606. 7 797 CPGWLDVLPF30.000 607. 8 648 VPQLGDTSPL 30.000 608. 9 530 APNNGTHGSL 20.000 609. 10577 CPHLQNSTQL 20.000 610. 11 164 FSMDGFRAEY 20.000 611. 12 240SSKEQNNPAW 15.000 612. 13 836 IARVRDVELL 13.500 613. 14 431 KPDQHFKPYL12.000 614. 15 441 TPDLPKRLHY 12.000 615. 16 280 GSFPSIYMPY 10.000 616.17 697 ASNRTSDSQY 10.000 617. 18 631 VSGFGKAMRM 10.000 618. 19 571ESLDCFCPHL 10.000 619. 20 746 VSGPIFDYNY 10.000 620. 21 219 ESHGIIDNNM10.000 621. 22 149 QSQCPEGFDL 7.500 622. 23 380 CNKMEYMTDY 6.000 623. 24637 AMRMPMWSSY 6.000 624. 25 407 RAHNIPHDFF 6.000 625. 26 760 DAPDEITKHL6.000 626. 28 444 LPKRLHYAKN 6.000 628. 29 340 KALQVVDHAF 6.000 629. 30120 LQKKDCCADY 6.000 630. 31 427 LSCRKPDQHF 5.000 631. 32 299 ISTLLKWLDL5.000 632. 33 130 KSVCQGETSW 5.000 633. 34 559 FSVCGFANPL 5.000 634. 35616 LQKNVDHCLL 4.500 635. 36 510 EPFENIEVYN 4.000 636. 37 723 KMWDYFHSVL4.000 637. 38 57 KCFDASFRGL 4.000 638. 39 700 RTSDSQYDAL 4.000 639. 40672 PPSESQKCSF 4.000 640. 41 188 KTCGIHSKYM 4.000 641. 42 88 TCVESTRIWM4.000 642. 43 411 IPHDFFSFNS 4.000 643. 44 568 LPTESLDCFC 4.000 644. 45774 VPIPTHYFVV 4.000 645. 46 310 KAERPRFYTM 3.600 646. 47 858 VSEILQLKTY3.000 647. 48 74 ACKDRGDGGW 3.000 648. 49 452 KNVRIDKVHL 3.000 649. 5023 IACIVLLALL 3.000 650.

TABLE XIX Motifs and Post-translational Modifications of 161P2F10BN-glycos ylation sites Number of matches: 10 1 236-239 NFSL (SEQ. ID.No. 701) 2 279-282 NGSF (SEQ. ID. No. 702) 3 290-293 NGSV (SEQ. ID. No.703) 4 426-429 NLSC (SEQ. ID. No. 704) 5 533-536 NGTH (SEQ. ID. No. 705)6 582-585 NSTQ (SEQ. ID. No. 706) 7 594-597 NLTQ (SEQ. ID. No. 707) 8687-690 NITH (SEQ. ID. No. 708) 9 699-702 NRTS (SEQ. ID. No. 709) 10789-792 NKSH (SEQ. ID. No. 710) cAMP-and cGMP-dependent protein kinasephosphory- lation site 14-17 KKNT (SEQ. ID. No. 711) Protein kinase Cphosphorylation sites Number of matches: 13 1 17-19 TLK 2 53-55 SCR 3428-430 SCR 4 62-64 SFR 5 92-94 STR 6 240-242 SSK 7 335-337 SAR 8 53-55SCR 9 428-430 SCR 10 502-504 SFK 11 603-605 TVK 12 676-678 SQK 13698-700 SNR Casein kinase II phosphorylation sites Number of matches: 151 88-91 TCVE (SEQ. ID. No. 712) 2 106-109 TRLE (SEQ. ID. No. 713) 3114-117 SCSD (SEQ. ID. No. 714) 4 138-141 SWLE (SEQ. ID. No. 715) 5240-243 SSKE (SEQ. ID. No. 716) 6 502-505 SFKE (SEQ. ID. No. 717) 7507-510 TEVE (SEQ. ID. No. 718) 8 551-554 SHAE (SEQ. ID. No. 719) 9584-587 TQLE (SEQ. ID. No. 720) 10 596-599 TQEE (SEQ. ID. No. 721) 11660-663 TVPD (SEQ. ID. No. 722) 12 704-707 SQYD (SEQ. ID. No. 723) 13813-816 TNVE (SEQ. ID. No. 724) 14 817-820 SCPE (SEQ. ID. No. 725) 15846-849 TGLD (SEQ. ID. No. 726) Tyrosine kinase phosphorylation site700-706 RTSDSQY (SEQ. ID. No. 727) N-myristoylation sites Number ofmatches: 11 1 38-43 GLGLGL (SEQ. ID. No. 728) 2 40-45 GLGLGL (SEQ. ID.No. 729) 3 38-43 GLGLGL (SEQ. ID. No. 730) 4 40-45 GLGLGL (SEQ. ID. No.731) 5 65-70 GLENCR (SEQ. ID. No. 732) 6 222-227 GIIDNN (SEQ. ID. No.733) 7 263-268 GLKAAT (SEQ. ID. No. 734) 8 273-278 GSEVAI (SEQ. ID. No.735) 9 280-285 GSFPSI (SEQ. ID. No. 736) 10 331-336 GGPVSA (SEQ. ID. No.737) 11 374-379 GMDQTY (SEQ. ID. No. 738) Cell attachment sequence 78-80RGD (SEQ. ID. No. 739) Somatomedin B domain signatures Number ofmatches: 2 1 69-89 CRCDVACKDRGDCCWDFEDTC (SEQ. ID. No. 740) 2 113-133CSCSDDCLQKKDCCADYKSVC (SEQ. ID. No. 741)

TABLE XX Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cvtochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide ig 19% Immunoglobulin domains areone hundred amino domain acids long and include a conserved intradomaindisulfide bond. WD40 18% WD domain, G-beta tandem repeats of about 40residues, repeat each containing a Trp-Asp motif. Function in signaltransduction and protein interaction PDZ 23% PDZ domain may function intargeting signaling molecules to sub-membranous sites LRR 28% LeucineRich Repeat short sequence motifs involved in protein-proteininteractions pkinase 23% Protein kinase domain conserved catalytic corecommon to both serine/threonine and tyrosine protein kinases containingan ATP binding site and a catalytic site PH 16% PH domain pleckstrinhomology involved in intracellular signaling or as constituents of thecytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in theextracellular domain of membrane- bound proteins or in secreted proteinsrvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ankrepeat Cytoplasmic protein, associates integral membrane proteins to thecytoskeleton oxidored_q1 32% NADH- membrane associated. InvolvedUbiquinone/plastoquin in proton translocation one (complex I), acrossthe membrane various chains efhand 24% EF hand calcium-binding domain,consists of a12 residue loop flanked on both sides by a 12 residuealpha-helical domain rvp 79% Retroviral aspartyl Aspartyl or acidproteases, protease centered on a catalytic aspartyl residue Collagen42% Collagen triple helix extracellular structural repeat (20 copies)proteins involved in formation of connective issue. The sequenceconsists of the G-X-Y and the polypeptide chains forms a triple helix.fn3 20% Fibronectin type III Located in the extracellular domainligand-binding region of receptors and is about 200 amino acid residueslong with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7transmembrane seven hydrophobic receptor (rhodopsin transmembraneregions, with family) the N-terminus located extracellularly while theC-terminus is cytoplasmic. Signal through G proteins

TABLE XXI Properties of 161P2F10B Bioinformatic Feature Program OutcomeORF (includes stop ORF finder 44-2671 codon) # of amino acids 875Transmembrane region TM Pred One TM, aa 23-41 HMMTop One TM, aa 23-45Sosui One TM, aa 23-45 TMHMM One TM, aa 23-45 Signal Peptide Signal Pnone pI pI/MW tool 6.12 Molecular weight pI/MW tool 100.09 kDaLocalization PSORT Plasma membrane 74% Golgi 30% PSORT II Endoplasmic30.4% Golgi 21.7% Motifs Pfam Somatomedin B, Type I phosphodiesterase/nucleotide pyrophosphatase Prints Cell Attachement RGD BlocksSomatomedin B, DNA/RNA non- specific endonuclease, Prosite Somatomedin B

1. A method of inhibiting growth of a solid tumor cell that expresses161P2F10B (SEQ ID NOS: 743 or 745), comprising: contacting said cellwith an antibody or fragment thereof that specifically binds to aprotein having an amino acid sequence of SEQ ID NOS: 743 or 745, whereinthe antibody or fragment thereof binds to the tumor cell via theprotein.
 2. The method of claim 1, wherein the antibody is a monoclonalantibody.
 3. The method of claim 1, wherein the monoclonal antibody isrecombinantly produced.
 4. The method of claim 3, wherein the antibodyis a single chain monoclonal antibody.
 5. The method of claim 1, whereinthe fragment thereof is selected from the group consisting of Fab,F(ab′)2, Fv and sFv fragment.
 6. The method of claim 1, wherein theantibody is a human antibody, a humanized antibody or a chimericantibody.
 7. The method of claim 1, wherein the antibody or fragmentthereof is labeled with an agent.
 8. The method of claim 7, wherein theagent is selected from the group consisting of radioactive isotopes,chemotherapeutic agents and toxins.
 9. The method of claim 8, whereinthe radioactive isotope is selected from the group consisting of ²¹¹At,¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P and radioactiveisotopes of Lu.
 10. The method of claim 9, wherein the chemotherapeuticagent is selected from the group consisting of taxol, actinomycin,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,gelonin, and calicheamicin.
 11. The method of claim 9, wherein the toxinis selected from the group consisting of diphtheria toxin, enomycin,phenomycin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain,mitogellin, modeccin A chain, and alpha-sarcin.